Enzymes having alpha-galactosidase activity and methods of use thereof

ABSTRACT

The invention relates to a-galactosidase and to polynucleotides encoding the α-galactosidase. In addition methods of designing new α-galactosidases and method of use thereof are also provided. The α-galactosidases have increased activity and stability at increased pH and temperature.

RELATED APPLICATIONS

[0001] This application is a divisional of and claims the benefit ofU.S. application Ser. No. 09/886,400, filed Jun. 20, 2001, which is acontinuation-in-part of U.S. application Ser. No. 09/619,072, filed Jul.19, 2000, now pending; which is a divisional of U.S. application Ser.No. 09/407,806, filed Sep. 28, 1999; which is a divisional of U.S.application Ser. No. 08/613,220, filed Mar. 8, 1996, now U.S. Pat. No.5,958,751, all of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

[0002] This invention relates generally to enzymes, polynucleotidesencoding the enzymes, the use of such polynucleotides and polypeptides,and more specifically to enzymes having alpha-galactosidase activity.

BACKGROUND

[0003] Alpha galactosidase is an enzyme which hydrolyses thenon-reducing terminal alpha 1-3,4,6 linked galactose from poly- andoligosaccharides. These saccharides are commonly found in legumes andare difficult to digest. As such, α-galactosidase has also been used asa digestive aid to break down raffinose, stachyose, and verbascose,found in such foods as beans and other gassy foods.

[0004] The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the invention is notentitled to antedate such disclosure by virtue of prior invention.

SUMMARY OF THE INVENTION

[0005] The invention provides an isolated nucleic acid having a sequenceas set forth in SEQ ID NO.:3 and variants thereof having at least 50%sequence identity to SEQ ID NO.:3 and encoding polypeptides havingalpha-galactosidase activity.

[0006] One aspect of the invention is an isolated nucleic acid having asequence as set forth in SEQ ID NO.:3, sequences substantially identicalthereto, and sequences complementary thereto.

[0007] Another aspect of the invention is an isolated nucleic acidincluding at least 10 consecutive bases of a sequence as set forth inSEQ ID NO.:3, sequences substantially identical thereto, and thesequences complementary thereto.

[0008] In yet another aspect, the invention provides an isolated nucleicacid encoding a polypeptide having a sequence as set forth in SEQ IDNO.:4 and variants thereof encoding a polypeptide havingalpha-galactosidase activity and having at least 50% sequence identityto such sequences.

[0009] Another aspect of the invention is an isolated nucleic acidencoding a polypeptide or a functional fragment thereof having asequence as set forth in SEQ ID NO.:4, and sequences substantiallyidentical thereto.

[0010] Another aspect of the invention is an isolated nucleic acidencoding a polypeptide having at least 10 consecutive amino acids of asequence as set forth in SEQ ID NO.:4, and sequences substantiallyidentical thereto.

[0011] In yet another aspect, the invention provides a purifiedpolypeptide having a sequence as set forth in SEQ ID NO.:4, andsequences substantially identical thereto.

[0012] Another aspect of the invention is an isolated or purifiedantibody that specifically binds to a polypeptide having a sequence asset forth in SEQ ID NO.:4, and sequences substantially identicalthereto.

[0013] Another aspect of the invention is an isolated or purifiedantibody or binding fragment thereof, which specifically binds to apolypeptide having at least 10 consecutive amino acids of one of thepolypeptides of SEQ ID NO.:4, and sequences substantially identicalthereto.

[0014] Another aspect of the invention is a method of making apolypeptide having a sequence as set forth in SEQ ID NO.:4, andsequences substantially identical thereto. The method includesintroducing a nucleic acid encoding the polypeptide into a host cell,wherein the nucleic acid is operably linked to a promoter, and culturingthe host cell under conditions that allow expression of the nucleicacid.

[0015] Another aspect of the invention is a method of making apolypeptide having at least 10 amino acids of a sequence as set forth inSEQ ID NO.:4, and sequences substantially identical thereto. The methodincludes introducing a nucleic acid encoding the polypeptide into a hostcell, wherein the nucleic acid is operably linked to a promoter, andculturing the host cell under conditions that allow expression of thenucleic acid, thereby producing the polypeptide.

[0016] Another aspect of the invention is a method of generating avariant including obtaining a nucleic acid having a sequence as setforth in SEQ ID NO.:3, sequences substantially identical thereto,sequences complementary to the sequences of SEQ ID NO.:3, fragmentscomprising at least 30 consecutive nucleotides of the foregoingsequences, and changing one or more nucleotides in the sequence toanother nucleotide, deleting one or more nucleotides in the sequence, oradding one or more nucleotides to the sequence.

[0017] Another aspect of the invention is a computer readable mediumhaving stored thereon a sequence as set forth in SEQ ID NO.:3, andsequences substantially identical thereto, or a polypeptide sequence asset forth in SEQ ID NO.:4, and sequences substantially identicalthereto.

[0018] Another aspect of the invention is a computer system including aprocessor and a data storage device wherein the data storage device hasstored thereon a sequence as set forth in SEQ ID NO.:3, and sequencessubstantially identical thereto, or a polypeptide having a sequence asset forth in SEQ ID NO.:4, and sequences substantially identicalthereto.

[0019] Another aspect of the invention is a method for comparing a firstsequence to a reference sequence wherein the first sequence is a nucleicacid having a sequence as set forth in SEQ ID NO.:3, and sequencessubstantially identical thereto, or a polypeptide code of SEQ ID NO.:4,and sequences substantially identical thereto. The method includesreading the first sequence and the reference sequence through use of acomputer program which compares sequences; and determining differencesbetween the first sequence and the reference sequence with the computerprogram.

[0020] Another aspect of the invention is a method for identifying afeature in a sequence as set forth in SEQ ID NO.:3, and sequencessubstantially identical thereto, or a polypeptide having a sequence asset forth in SEQ ID NO.:4, and sequences substantially identicalthereto, including reading the sequence through the use of a computerprogram which identifies features in sequences; and identifying featuresin the sequence with the computer program.

[0021] Another aspect of the invention is an assay for identifyingfragments or variants of SEQ ID NO.:4, and sequences substantiallyidentical thereto, which retain the enzymatic function of thepolypeptides of SEQ ID NO.:4, and sequences substantially identicalthereto. The assay includes contacting the polypeptide of SEQ ID NO.:4,sequences substantially identical thereto, or polypeptide fragment orvariant with a substrate molecule under conditions which allow thepolypeptide fragment or variant to function, and detecting either adecrease in the level of substrate or an increase in the level of thespecific reaction product of the reaction between the polypeptide andsubstrate thereby identifying a fragment or variant of such sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0023]FIG. 1 is a block diagram of a computer system.

[0024]FIG. 2 is a flow diagram illustrating one embodiment of a processfor comparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

[0025]FIG. 3 is a flow diagram illustrating one embodiment of a processin a computer for determining whether two sequences are homologous.

[0026]FIG. 4 is a flow diagram illustrating one embodiment of anidentifier process 300 for detecting the presence of a feature in asequence.

[0027]FIG. 5 is an illustration of the full-length DNA and correspondingdeduced amino acid sequence of Thernococcus alcaliphilus AEDII12RAα-galactosidase 18GC of the present invention. Sequencing was performedusing a 378 automated DNA sequencer (Applied Biosystems, Inc.).

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention relates to α-galactosidase andpolynucleotides encoding it. As used herein, the term “α-galactosidase”encompasses enzymes having α-galactosidase activity.

[0029] The polynucleotides of the invention have been identified asencoding polypeptides having α-galactosidase activity.

[0030] Definitions

[0031] The phrases “nucleic acid” or “nucleic acid sequence” as usedherein refer to an oligonucleotide, nucleotide, polynucleotide, or to afragment of any of these, to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent asense or antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material, natural or synthetic in origin.

[0032] A “coding sequence of” or a “nucleotide sequence encoding” aparticular polypeptide or protein, is a nucleic acid sequence which istranscribed and translated into a polypeptide or protein when placedunder the control of appropriate regulatory sequences.

[0033] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as, where applicable,intervening sequences (introns) between individual coding segments(exons).

[0034] “Amino acid” or “amino acid sequence” as used herein refer to anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these, and to naturallyoccurring or synthetic molecules.

[0035] The term “polypeptide” as used herein, refers to amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain modified amino acids other than the20 gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Modificationscan occur anywhere in the polypeptide, including the peptide backbone,the amino acid side-chains and the amino or carboxyl termini. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide. Also agiven polypeptide may have many types of modifications. Modificationsinclude acetylation, acylation, ADP-ribosylation, amidation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment of aphosphytidylinositol, cross-linking cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristolyation, oxidation, pergylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, and transfer-RNA mediated addition of aminoacids to protein such as arginylation. (See Creighton, T. E.,Proteins—Structure and Molecular Properties 2nd Ed., W. H. Freeman andCompany, New York (1993); Posttranslational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12(1983)).

[0036] As used herein, the term “isolated” means that the material isremoved from its original environment (e.g., the natural environment ifit is naturally occurring). For example, a naturally-occurringpolynucleotide or polypeptide present in a living animal is notisolated, but the same polynucleotide or polypeptide, separated fromsome or all of the coexisting materials in the natural system, isisolated. Such polynucleotides could be part of a vector and/or suchpolynucleotides or polypeptides could be part of a composition, andstill be isolated in that such vector or composition is not part of itsnatural environment.

[0037] As used herein, the term “purified” does not require absolutepurity; rather, it is intended as a relative definition. Individualnucleic acids obtained from a library have been conventionally purifiedto electrophoretic homogeneity. The sequences obtained from these clonescould not be obtained directly either from the library or from totalhuman DNA. The purified nucleic acids of the invention have beenpurified from the remainder of the genomic DNA in the organism by atleast 10⁴-10⁶ fold. However, the term “purified” also includes nucleicacids which have been purified from the remainder of the genomic DNA orfrom other sequences in a library or other environment by at least oneorder of magnitude, typically two or three orders, and more typicallyfour or five orders of magnitude.

[0038] As used herein, the term “recombinant” means that the nucleicacid is adjacent to a “backbone” nucleic acid to which it is notadjacent in its natural environment. Additionally, to be “enriched” thenucleic acids will represent 5% or more of the number of nucleic acidinserts in a population of nucleic acid backbone molecules. Backbonemolecules according to the invention include nucleic acids such asexpression vectors, self-replicating nucleic acids, viruses, integratingnucleic acids, and other vectors or nucleic acids used to maintain ormanipulate a nucleic acid insert of interest. Typically, the enrichednucleic acids represent 15% or more of the number of nucleic acidinserts in the population of recombinant backbone molecules. Moretypically, the enriched nucleic acids represent 50% or more of thenumber of nucleic acid inserts in the population of recombinant backbonemolecules. In a one embodiment, the enriched nucleic acids represent 90%or more of the number of nucleic acid inserts in the population ofrecombinant backbone molecules.

[0039] “Recombinant” polypeptides or proteins refer to polypeptides orproteins produced by recombinant DNA techniques; i.e., produced fromcells transformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or protein are thoseprepared by chemical synthesis. Solid-phase chemical peptide synthesismethods can also be used to synthesize the polypeptide or fragments ofthe invention. Such method have been known in the art since the early1960's (Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (Seealso Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2ndEd., Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have recentlybeen employed in commercially available laboratory peptide design andsynthesis kits (Cambridge Research Biochemicals). Such commerciallyavailable laboratory kits have generally utilized the teachings of H. M.Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and providefor synthesizing peptides upon the tips of a multitude of “rods” or“pins” all of which are connected to a single plate. When such a systemis utilized, a plate of rods or pins is inverted and inserted into asecond plate of corresponding wells or reservoirs, which containsolutions for attaching or anchoring an appropriate amino acid to thepin's or rod's tips. By repeating such a process step, i.e., invertingand inserting the rod's and pin's tips into appropriate solutions, aminoacids are built into desired peptides. In addition, a number ofavailable FMOC peptide synthesis systems are available. For example,assembly of a polypeptide or fragment can be carried out on a solidsupport using an Applied Biosystems, Inc. Model 43 1A automated peptidesynthesizer. Such equipment provides ready access to the peptides of theinvention, either by direct synthesis or by synthesis of a series offragments that can be coupled using other known techniques.

[0040] A promoter sequence is “operably linked to” a coding sequencewhen RNA polymerase which initiates transcription at the promoter willtranscribe the coding sequence into mRNA.

[0041] “Plasmids” are designated by a lower case “p” preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed herein are known in the art and will be apparent to theordinarily skilled artisan.

[0042] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion, gel electrophoresis may beperformed to isolate the desired fragment.

[0043] “Oligonucleotide” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0044] The phrase “substantially identical” in the context of twonucleic acids or polypeptides, refers to two or more sequences that haveat least 50%, 60%, 70%, 80%, and in some aspects 90-95% nucleotide oramino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using one of the known sequence comparisonalgorithms or by visual inspection. Typically, the substantial identityexists over a region of at least about 100 residues, and most commonlythe sequences are substantially identical over at least about 150-200residues. In some embodiments, the sequences are substantially identicalover the entire length of the coding regions.

[0045] Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site of the molecule, and provided that thepolypeptide essentially retains its functional properties. Aconservative amino acid substitution, for example, substitutes one aminoacid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucin, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid or glutamine for asparagine). One or more amino acids canbe deleted, for example, from an α-galactosidase polypeptide, resultingin modification of the structure of the polypeptide, withoutsignificantly altering its biological activity. For example, amino- orcarboxyl-terminal amino acids that are not required for α-galactosidasebiological activity can be removed. Modified polypeptide sequences ofthe invention can be assayed for a-galactosidase biological activity byany number of methods, including contacting the modified polypeptidesequence with an α-galactosidase substrate and determining whether themodified polypeptide decreases the amount of specific substrate in theassay or increases the bioproducts of the enzymatic reaction of afunctional α-galactosidase polypeptide with the substrate.

[0046] “Fragments” as used herein are a portion of a naturally occurringprotein which can exist in at least two different conformations.Fragments can have the same or substantially the same amino acidsequence as the naturally occurring protein. “Substantially the same”means that an amino acid sequence is largely, but not entirely, thesame, but retains at least one functional activity of the sequence towhich it is related. In general two amino acid sequences are“substantially the same” or “substantially homologous” if they are atleast about 85% identical. Fragments which have different threedimensional structures as the naturally occurring protein are alsoincluded. An example of this, is a “pro-form” molecule, such as a lowactivity proprotein that can be modified by cleavage to produce a matureenzyme with significantly higher activity.

[0047] “Hybridization” refers to the process by which a nucleic acidstrand joins with a complementary strand through base pairing.Hybridization reactions can be sensitive and selective so that aparticular sequence of interest can be identified even in samples inwhich it is present at low concentrations. Suitably stringent conditionscan be defined by, for example, the concentrations of salt or formamidein the prehybridization and hybridization solutions, or by thehybridization temperature, and are well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

[0048] For example, hybridization under high stringency conditions couldoccur in about 50% formamide at about 37° C. to 42° C. Hybridizationcould occur under reduced stringency conditions in about 35% to 25%formamide at about 30° C. to 35° C. In particular, hybridization couldoccur under high stringency conditions at 42° C. in 50% formamide, 5XSSPE, 0.3% SDS, and 200 n/ml sheared and denatured salmon sperm DNA.Hybridization could occur under reduced stringency conditions asdescribed above, but in 35% formamide at a reduced temperature of 35° C.The temperature range corresponding to a particular level of stringencycan be further narrowed by calculating the purine to pyrimidine ratio ofthe nucleic acid of interest and adjusting the temperature accordingly.Variations on the above ranges and conditions are well known in the art.

[0049] The term “variant” refers to polynucleotides or polypeptides ofthe invention modified at one or more base pairs, codons, introns,exons, or amino acid residues (respectively) yet still retain thebiological activity of an cc-galactosidase of the invention. Variantscan be produced by any number of means included methods such as, forexample, error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSMand any combination thereof.

[0050] Enzymes are highly selective catalysts. Their hallmark is theability to catalyze reactions with exquisite stereo-, regio-, and chemo-selectivities that are unparalleled in conventional synthetic chemistry.Moreover, enzymes are remarkably versatile. They can be tailored tofunction in organic solvents, operate at extreme pHs (for example, highpHs and low pHs) extreme temperatures (for example, high temperaturesand low temperatures), extreme salinity levels (for example, highsalinity and low salinity), and catalyze reactions with compounds thatare structurally unrelated to their natural, physiological substrates.

[0051] Enzymes are reactive toward a wide range of natural and unnaturalsubstrates, thus enabling the modification of virtually any organic leadcompound. Moreover, unlike traditional chemical catalysts, enzymes arehighly enantio- and regio-selective. The high degree of functional groupspecificity exhibited by enzymes enables one to keep track of eachreaction in a synthetic sequence leading to a new active compound.Enzymes are also capable of catalyzing many diverse reactions unrelatedto their physiological function in nature. For example, peroxidasescatalyze the oxidation of phenols by hydrogen peroxide. Peroxidases canalso catalyze hydroxylation reactions that are not related to the nativefunction of the enzyme. Other examples are proteases which catalyze thebreakdown of polypeptides. In organic solution some proteases can alsoacylate sugars, a function unrelated to the native function of theseenzymes.

[0052] The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds.

[0053] Each biocatalyst is specific for one functional group, or severalrelated functional groups, and can react with many starting compoundscontaining this functional group.

[0054] The biocatalytic reactions produce a population of derivativesfrom a single starting compound. These derivatives can be subjected toanother round of biocatalytic reactions to produce a second populationof derivative compounds. Thousands of variations of the originalcompound can be produced with each iteration of biocatalyticderivatization.

[0055] Enzymes react at specific sites of a starting compound withoutaffecting the rest of the molecule, a process which is very difficult toachieve using traditional chemical methods. This high degree ofbiocatalytic specificity provides the means to identify a single activecompound within the library. The library is characterized by the seriesof biocatalytic reactions used to produce it, a so-called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies, and compounds can besynthesized and tested free in solution using virtually any type ofscreening assay. It is important to note, that the high degree ofspecificity of enzyme reactions on functional groups allows for the“tracking” of specific enzymatic reactions that make up thebiocatalytically produced library.

[0056] Many of the procedural steps are performed using roboticautomation enabling the execution of many thousands of biocatalyticreactions and screening assays per day as well as ensuring a high levelof accuracy and reproducibility. As a result, a library of derivativecompounds can be produced in a matter of weeks which would take years toproduce using current chemical methods. (For further teachings onmodification of molecules, including small molecules, seePCTIUS94/09174, herein incorporated by reference in its entirety).

[0057] In one aspect, the present invention provides a non-stochasticmethod termed synthetic gene reassembly, that is somewhat related tostochastic shuffling, save that the nucleic acid building blocks are notshuffled or concatenated or chimerized randomly, but rather areassembled non-stochastically.

[0058] The SLR method does not depend on the presence of a high level ofhomology between polynucleotides to be shuffled. The invention can beused to non-stochastically generate libraries (or sets) of progenymolecules comprised of over 10¹⁰⁰ different chimeras. Conceivably, SLRcan even be used to generate libraries comprised of over 10¹⁰⁰⁰different progeny chimeras.

[0059] Thus, in one aspect, the invention provides a non-stochasticmethod of producing a set of finalized chimeric nucleic acid moleculeshaving an overall assembly order that is chosen by design, which methodis comprised of the steps of generating by design a plurality ofspecific nucleic acid building blocks having serviceable mutuallycompatible ligatable ends, and assembling these nucleic acid buildingblocks, such that a designed overall assembly order is achieved.

[0060] The mutually compatible ligatable ends of the nucleic acidbuilding blocks to be assembled are considered to be “serviceable” forthis type of ordered assembly if they enable the building blocks to becoupled in predetermined orders. Thus, in one aspect, the overallassembly order in which the nucleic acid building blocks can be coupledis specified by the design of the ligatable ends and, if more than oneassembly step is to be used, then the overall assembly order in whichthe nucleic acid building blocks can be coupled is also specified by thesequential order of the assembly step(s). In a one embodiment of theinvention, the annealed building pieces are treated with an enzyme, suchas a ligase (e.g., T4 DNA ligase) to achieve covalent bonding of thebuilding pieces.

[0061] In a another embodiment, the design of nucleic acid buildingblocks is obtained upon analysis of the sequences of a set of progenitornucleic acid templates that serve as a basis for producing a progeny setof finalized chimeric nucleic acid molecules. These progenitor nucleicacid templates thus serve as a source of sequence information that aidsin the design of the nucleic acid building blocks that are to bemutagenized, i.e. chimerized or shuffled.

[0062] In one exemplification, the invention provides for thechimerization of a family of related genes and their encoded family ofrelated products. In a particular exemplification, the encoded productsare enzymes. The α-galactosidase of the present invention can bemutagenized in accordance with the methods described herein.

[0063] Thus according to one aspect of the invention, the sequences of aplurality of progenitor nucleic acid templates (e.g., the polynucleotideof SEQ ID NO.:3) are aligned in order to select one or more demarcationpoints, which demarcation points can be located at an area of homology.The demarcation points can be used to delineate the boundaries ofnucleic acid building blocks to be generated. Thus, the demarcationpoints identified and selected in the progenitor molecules serve aspotential chimerization points in the assembly of the progeny molecules.

[0064] Typically a serviceable demarcation point is an area of homology(comprised of at least one homologous nucleotide base) shared by atleast two progenitor templates, but the demarcation point can be an areaof homology that is shared by at least half of the progenitor templates,at least two thirds of the progenitor templates, at least three fourthsof the progenitor templates, and preferably at almost all of theprogenitor templates. Even more preferably still a serviceabledemarcation point is an area of homology that is shared by all of theprogenitor templates.

[0065] In a one embodiment, the gene reassembly process is performedexhaustively in order to generate an exhaustive library. In other words,all possible ordered combinations of the nucleic acid building blocksare represented in the set of finalized chimeric nucleic acid molecules.At the same time, the assembly order (i.e. the order of assembly of eachbuilding block in the 5′ to 3 sequence of each finalized chimericnucleic acid) in each combination is by design (or non-stochastic).Because of the non-stochastic nature of the method, the possibility ofunwanted side products is greatly reduced.

[0066] In another embodiment, the method provides that the genereassembly process is performed systematically, for example to generatea systematically compartmentalized library, with compartments that canbe screened systematically, e.g., one by one. In other words theinvention provides that, through the selective and judicious use ofspecific nucleic acid building blocks, coupled with the selective andjudicious use of sequentially stepped assembly reactions, anexperimental design can be achieved where specific sets of progenyproducts are made in each of several reaction vessels. This allows asystematic examination and screening procedure to be performed. Thus, itallows a potentially very large number of progeny molecules to beexamined systematically in smaller groups.

[0067] Because of its ability to perform chimerizations in a manner thatis highly flexible yet exhaustive and systematic as well, particularlywhen there is a low level of homology among the progenitor molecules,the instant invention provides for the generation of a library (or set)comprised of a large number of progeny molecules. Because of thenon-stochastic nature of the instant gene reassembly invention, theprogeny molecules generated preferably comprise a library of finalizedchimeric nucleic acid molecules having an overall assembly order that ischosen by design. In a particularly embodiment, such a generated libraryis comprised of greater than 10³ to greater than 10¹⁰⁰⁰ differentprogeny molecular species.

[0068] In one aspect, a set of finalized chimeric nucleic acidmolecules, produced as described is comprised of a polynucleotideencoding a polypeptide. According to one embodiment, this polynucleotideis a gene, which may be a man-made gene. According to anotherembodiment, this polynucleotide is a gene pathway, which may be aman-made gene pathway. The invention provides that one or more man-madegenes generated by the invention may be incorporated into a man-madegene pathway, such as pathway operable in a eukaryotic organism(including a plant).

[0069] In another exemplification, the synthetic nature of the step inwhich the building blocks are generated allows the design andintroduction of nucleotides (e.g., one or more nucleotides, which maybe, for example, codons or introns or regulatory sequences) that canlater be optionally removed in an in vitro process (e.g., bymutagenesis) or in an in vivo process (e.g., by utilizing the genesplicing ability of a host organism). It is appreciated that in manyinstances the introduction of these nucleotides may also be desirablefor many other reasons in addition to the potential benefit of creatinga serviceable demarcation point.

[0070] Thus, according to another embodiment, the invention providesthat a nucleic acid building block can be used to introduce an intron.Thus, the invention provides that functional introns may be introducedinto a man-made gene of the invention. The invention also provides thatfunctional introns may be introduced into a man-made gene pathway of theinvention. Accordingly, the invention provides for the generation of achimeric polynucleotide that is a man-made gene containing one (or more)artificially introduced intron(s).

[0071] Accordingly, the invention also provides for the generation of achimeric polynucleotide that is a man-made gene pathway containing one(or more) artificially introduced intron(s). Preferably, theartificially introduced intron(s) are functional in one or more hostcells for gene splicing much in the way that naturally-occurring intronsserve functionally in gene splicing. The invention provides a process ofproducing man-made intron-containing polynucleotides to be introducedinto host organisms for recombination and/or splicing.

[0072] A man-made gene produced using the invention can also serve as asubstrate for recombination with another nucleic acid. Likewise, aman-made gene pathway produced using the invention can also serve as asubstrate for recombination with another nucleic acid. In a preferredinstance, the recombination is facilitated by, or occurs at, areas ofhomology between the man-made, intron-containing gene and a nucleicacid, which serves as a recombination partner. In a particularlypreferred instance, the recombination partner may also be a nucleic acidgenerated by the invention, including a man-made gene or a man-made genepathway. Recombination may be facilitated by or may occur at areas ofhomology that exist at the one (or more) artificially introducedintron(s) in the man-made gene.

[0073] The synthetic gene reassembly method of the invention utilizes aplurality of nucleic acid building blocks, each of which preferably hastwo ligatable ends. The two ligatable ends on each nucleic acid buildingblock may be two blunt ends (i.e. each having an overhang of zeronucleotides), or preferably one blunt end and one overhang, or morepreferably still two overhangs.

[0074] A useful overhang for this purpose may be a 3′ overhang or a 5′overhang. Thus, a nucleic acid building block may have a 3′ overhang oralternatively a 5′ overhang or alternatively two 3′ overhangs oralternatively two 5′ overhangs. The overall order in which the nucleicacid building blocks are assembled to form a finalized chimeric nucleicacid molecule is determined by purposeful experimental design and is notrandom.

[0075] According to one preferred embodiment, a nucleic acid buildingblock is generated by chemical synthesis of two single-stranded nucleicacids (also referred to as single-stranded oligos) and contacting themso as to allow them to anneal to form a double-stranded nucleic acidbuilding block.

[0076] A double-stranded nucleic acid building block can be of variablesize. The sizes of these building blocks can be small or large.Preferred sizes for building block range from 1 base pair (not includingany overhangs) to 100,000 base pairs (not including any overhangs).Other preferred size ranges are also provided, which have lower limitsof from 1 bp to 10,000 bp (including every integer value in between),and upper limits of from 2 bp to 100,000 bp (including every integervalue in between).

[0077] Many methods exist by which a double-stranded nucleic acidbuilding block can be generated that is serviceable for the invention;and these are known in the art and can be readily performed by theskilled artisan.

[0078] According to one embodiment, a double-stranded nucleic acidbuilding block is generated by first generating two single strandednucleic acids and allowing them to anneal to form a double-strandednucleic acid building block. The two strands of a double-strandednucleic acid building block may be complementary at every nucleotideapart from any that form an overhang; thus containing no mismatches,apart from any overhang(s). According to another embodiment, the twostrands of a double-stranded nucleic acid building block arecomplementary at fewer than every nucleotide apart from any that form anoverhang. Thus, according to this embodiment, a double-stranded nucleicacid building block can be used to introduce codon degeneracy.Preferably the codon degeneracy is introduced using the site-saturationmutagenesis described herein, using one or more N,N,G/T cassettes oralternatively using one or more N,N,N cassettes.

[0079] The in vivo recombination method of the invention can beperformed blindly on a pool of unknown hybrids or alleles of a specificpolynucleotide or sequence. However, it is not necessary to know theactual DNA or RNA sequence of the specific polynucleotide.

[0080] The approach of using recombination within a mixed population ofgenes can be useful for the generation of any useful proteins, forexample, interleukin I, antibodies, tPA and growth hormone. Thisapproach may be used to generate proteins having altered specificity oractivity. The approach may also be useful for the generation of hybridnucleic acid sequences, for example, promoter regions, introns, exons,enhancer sequences, 31 untranslated regions or 51 untranslated regionsof genes. Thus this approach may be used to generate genes havingincreased rates of expression. This approach may also be useful in thestudy of repetitive DNA sequences. Finally, this approach may be usefulto mutate ribozymes or aptamers.

[0081] In one aspect the invention described herein is directed to theuse of repeated cycles of reductive reassortment, recombination andselection which allow for the directed molecular evolution of highlycomplex linear sequences, such as DNA, RNA or proteins thoroughrecombination.

[0082] In vivo shuffling of molecules is useful in providing variantsand can be performed utilizing the natural property of cells torecombine multimers. While recombination in vivo has provided the majornatural route to molecular diversity, genetic recombination remains arelatively complex process that involves 1) the recognition ofhomologies; 2) strand cleavage, strand invasion, and metabolic stepsleading to the production of recombinant chiasma; and finally 3) theresolution of chiasma into discrete recombined molecules. The formationof the chiasma requires the recognition of homologous sequences.

[0083] In another embodiment, the invention includes a method forproducing a hybrid polynucleotide from at least a first polynucleotideand a second polynucleotide. The invention can be used to produce ahybrid polynucleotide by introducing at least a first polynucleotide anda second polynucleotide which share at least one region of partialsequence homology into a suitable host cell. The regions of partialsequence homology promote processes which result in sequencereorganization producing a hybrid polynucleotide. The term “hybridpolynucleotide”, as used herein, is any nucleotide sequence whichresults from the method of the present invention and contains sequencefrom at least two original polynucleotide sequences. Such hybridpolynucleotides can result from intermolecular recombination eventswhich promote sequence integration between DNA molecules. In addition,such hybrid polynucleotides can result from intramolecular reductivereassortment processes which utilize repeated sequences to alter anucleotide sequence within a DNA molecule.

[0084] The invention provides a means for generating hybridpolynucleotides which may encode biologically active hybrid polypeptides(e.g., hybrid α-galactosidase). In one aspect, the originalpolynucleotides encode biologically active polypeptides. The method ofthe invention produces new hybrid polypeptides by utilizing cellularprocesses which integrate the sequence of the original polynucleotidessuch that the resulting hybrid polynucleotide encodes a polypeptidedemonstrating activities derived from the original biologically activepolypeptides. For example, the original polynucleotides may encode aparticular enzyme from different microorganisms. An enzyme encoded by afirst polynucleotide from one organism or variant may, for example,function effectively under a particular environmental condition, e.g.high salinity. An enzyme encoded by a second polynucleotide from adifferent organism or variant may function effectively under a differentenvironmental condition, such as extremely high temperatures. A hybridpolynucleotide containing sequences from the first and second originalpolynucleotides may encode an enzyme which exhibits characteristics ofboth enzymes encoded by the original polynucleotides. Thus, the enzymeencoded by the hybrid polynucleotide may function effectively underenvironmental conditions shared by each of the enzymes encoded by thefirst and second polynucleotides, e.g., high salinity and extremetemperatures.

[0085] Enzymes encoded by the polynucleotides of the invention include,but are not limited to, hydrolases, such as α-galactosidases. A hybridpolypeptide resulting from the method of the invention may exhibitspecialized enzyme activity not displayed in the original enzymes. Forexample, following recombination and/or reductive reassortment ofpolynucleotides encoding hydrolase activities, the resulting hybridpolypeptide encoded by a hybrid polynucleotide can be screened forspecialized hydrolase activities obtained from each of the originalenzymes, i.e. the type of bond on which the hydrolase acts and thetemperature at which the hydrolase functions. Thus, for example, thehydrolase may be screened to ascertain those chemical functionalitieswhich distinguish the hybrid hydrolase from the original hydrolases,such as:(a) amide (peptide bonds), i.e., proteases; (b) ester bonds,i.e., esterases and lipases; (c) acetals, i.e., glycosidases and, forexample, the temperature, pH or salt concentration at which the hybridpolypeptide functions.

[0086] Sources of the original polynucleotides may be isolated fromindividual organisms (“isolates”), collections of organisms that havebeen grown in defined media (“enrichment cultures”), or, uncultivatedorganisms (“environmental samples”). The use of a culture-independentapproach to derive polynucleotides encoding novel bioactivities fromenvironmental samples is most preferable since it allows one to accessuntapped resources of biodiversity.

[0087] “Environmental libraries” are generated from environmentalsamples and represent the collective genomes of naturally occurringorganisms archived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample which may be under-represented by several orders of magnitudecompared to the dominant species.

[0088] For example, gene libraries generated from one or moreuncultivated microorganisms are screened for an activity of interest.Potential pathways encoding bioactive molecules of interest are firstcaptured in prokaryotic cells in the form of gene expression libraries.Polynucleotides encoding activities of interest are isolated from suchlibraries and introduced into a host cell. The host cell is grown underconditions which promote recombination and/or reductive reassortmentcreating potentially active biomolecules with novel or enhancedactivities.

[0089] The microorganisms from which the polynucleotide may be preparedinclude prokaryotic microorganisms, such as Eubacteria andArchaebacteria, and lower eukaryotic microorganisms such as fungi, somealgae and protozoa. Polynucleotides may be isolated from environmentalsamples in which case the nucleic acid may be recovered withoutculturing of an organism or recovered from one or more culturedorganisms. In one aspect, such microorganisms may be extremophiles, suchas hyperthermophiles, psychrophiles, psychrotrophs, halophiles,barophiles and acidophiles. Polynucleotides encoding enzymes isolatedfrom extremophilic microorganisms are particularly preferred. Suchenzymes may function at temperatures above 100° C. in terrestrial hotsprings and deep sea thermal vents, at temperatures below 0° C. inarctic waters, in the saturated salt environment of the Dead Sea, at pHvalues around 0 in coal deposits and geothermal sulfur-rich springs, orat pH values greater than 11 in sewage sludge. For example, severalesterases and lipases cloned and expressed from extremophilic organismsshow high activity throughout a wide range of temperatures and pHs.

[0090] Polynucleotides selected and isolated as hereinabove describedare introduced into a suitable host cell. A suitable host cell is anycell which is capable of promoting recombination and/or reductivereassortment. The selected polynucleotides are preferably already in avector which includes appropriate control sequences. The host cell canbe a higher eukaryotic cell, such as a mammalian cell, or a lowereukaryotic cell, such as a yeast cell, or preferably, the host cell canbe a prokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis et al., 1986).

[0091] As representative examples of appropriate hosts, there may bementioned:bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowesmelanoma; adenoviruses; and plant cells. The selection of an appropriatehost is deemed to be within the scope of those skilled in the art fromthe teachings herein.

[0092] With particular references to various mammalian cell culturesystems that can be employed to express recombinant protein, examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described in “SV40-transformed simian cells support thereplication of early SV40 mutants” (Gluzman, 1981), and other cell linescapable of expressing a compatible vector, for example, the C127, 3T3,CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprisean origin of replication, a suitable promoter and enhancer, and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0093] Host cells containing the polynucleotides of interest can becultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transformants or amplifying genes. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan. The clones which areidentified as having the specified enzyme activity may then be sequencedto identify the polynucleotide sequence encoding an enzyme having theenhanced activity.

[0094] In another aspect, it is envisioned the method of the presentinvention can be used to generate novel polynucleotides encodingbiochemical pathways from one or more operons or gene clusters orportions thereof. For example, bacteria and many eukaryotes have acoordinated mechanism for regulating genes whose products are involvedin related processes. The genes are clustered, in structures referred toas “gene clusters,” on a single chromosome and are transcribed togetherunder the control of a single regulatory sequence, including a singlepromoter which initiates transcription of the entire cluster. Thus, agene cluster is a group of adjacent genes that are either identical orrelated, usually as to their function. An example of a biochemicalpathway encoded by gene clusters are polyketides. Polyketides aremolecules which are an extremely rich source of bioactivities, includingantibiotics (such as tetracyclines and erythromycin), anti-cancer agents(daunomycin), immunosuppressants (FK506 and rapamycin), and veterinaryproducts (monensin). Many polyketides (produced by polyketide synthases)are valuable as therapeutic agents. Polyketide synthases aremultifunctional enzymes that catalyze the biosynthesis of an enormousvariety of carbon chains differing in length and patterns offunctionality and cyclization. Polyketide synthase genes fall into geneclusters and at least one type (designated type I) of polyketidesynthases have large size genes and enzymes, complicating geneticmanipulation and in vitro studies of these genes/proteins.

[0095] Gene cluster DNA can be isolated from different organisms andligated into vectors, particularly vectors containing expressionregulatory sequences which can control and regulate the production of adetectable protein or protein-related array activity from the ligatedgene clusters. Use of vectors which have an exceptionally large capacityfor exogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E. coliis a plasmid which affect high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples. Aparticularly preferred embodiment is to use cloning vectors, referred toas “fosmids” or bacterial artificial chromosome (BAC) vectors. These arederived from E. coli f-factor which is able to stably integrate largesegments of genomic DNA. When integrated with DNA from a mixeduncultured environmental sample, this makes it possible to achieve largegenomic fragments in the form of a stable “environmental DNA library.”Another type of vector for use in the present invention is a cosmidvector. Cosmid vectors were originally designed to clone and propagatelarge segments of genomic DNA. Cloning into cosmid vectors is describedin detail in Sambrook et al., Molecular Cloning:A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press (1989). Once ligated into anappropriate vector, two or more vectors containing different polyketidesynthase gene clusters can be introduced into a suitable host cell.Regions of partial sequence homology shared by the gene clusters willpromote processes which result in sequence reorganization resulting in ahybrid gene cluster. The novel hybrid gene cluster can then be screenedfor enhanced activities not found in the original gene clusters.

[0096] Therefore, in a one embodiment, the invention relates to a methodfor producing a biologically active hybrid polypeptide and screeningsuch a polypeptide for enhanced activity by:

[0097] 1) introducing at least a first polynucleotide in operablelinkage and a second polynucleotide in operable linkage, said at leastfirst polynucleotide and second polynucleotide sharing at least oneregion of partial sequence homology, into a suitable host cell;

[0098] 2) growing the host cell under conditions which promote sequencereorganization resulting in a hybrid polynucleotide in operable linkage;

[0099] 3) expressing a hybrid polypeptide encoded by the hybridpolynucleotide;

[0100] 4) screening the hybrid polypeptide under conditions whichpromote identification of enhanced biological activity; and

[0101] 5) isolating the a polynucleotide encoding the hybridpolypeptide.

[0102] Methods for screening for various enzyme activities are known tothose of skill in the art and are discussed throughout the presentspecification. Such methods may be employed when isolating thepolypeptides and polynucleotides of the invention.

[0103] As representative examples of expression vectors which may beused, there may be mentioned viral particles, baculovirus, phage,plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes,viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, aspergillus and yeast).Thus, for example, the DNA may be included in any one of a variety ofexpression vectors for expressing a polypeptide. Such vectors includechromosomal, nonchromosomal and synthetic DNA sequences. Large numbersof suitable vectors are known to those of skill in the art, and arecommercially available. The following vectors are provided by way ofexample; Bacterial:pQE vectors (Qiagen), pBluescript plasmids, pNHvectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540,pRIT2T (Pharmacia); Eukaryotic:pXT1, pSG5 (Stratagene), pSVK3, pBPV,pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vectormay be used so long as they are replicable and viable in the host. Lowcopy number or high copy number vectors may be employed with the presentinvention.

[0104] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directRNA synthesis. Particular named bacterial promoters include lacI, lacZ,T3, T7, gpt, lambda P_(R), P_(L) and trp. Eukaryotic promoters includeCMV immediate early, HSV thymidine kinase, early and late SV40, LTRsfrom retrovirus, and mouse metallothionein-I. Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art. The expression vector also contains a ribosome bindingsite for translation initiation and a transcription terminator. Thevector may also include appropriate sequences for amplifying expression.Promoter regions can be selected from any desired gene usingchloramphenicol transferase (CAT) vectors or other vectors withselectable markers. In addition, the expression vectors preferablycontain one or more selectable marker genes to provide a phenotypictrait for selection of transformed host cells such as dihydrofolatereductase or neomycin resistance for eukaryotic cell culture, or such astetracycline or ampicillin resistance in E. coli.

[0105] In vivo reassortment is focused on “inter-molecular” processescollectively referred to as “recombination” which in bacteria, isgenerally viewed as a “RecA-dependent” phenomenon. The invention canrely on recombination processes of a host cell to recombine andre-assort sequences, or the cells' ability to mediate reductiveprocesses to decrease the complexity of quasi-repeated sequences in thecell by deletion. This process of “reductive reassortment” occurs by an“intra-molecular”, RecA-independent process.

[0106] Therefore, in another aspect of the invention, novelpolynucleotides can be generated by the process of reductivereassortment. The method involves the generation of constructscontaining consecutive sequences (original encoding sequences), theirinsertion into an appropriate vector, and their subsequent introductioninto an appropriate host cell. The reassortment of the individualmolecular identities occurs by combinatorial processes between theconsecutive sequences in the construct possessing regions of homology,or between quasi-repeated units. The reassortment process recombinesand/or reduces the complexity and extent of the repeated sequences, andresults in the production of novel molecular species. Various treatmentsmay be applied to enhance the rate of reassortment. These could includetreatment with ultra-violet light, or DNA damaging chemicals, and/or theuse of host cell lines displaying enhanced levels of “geneticinstability”. Thus the reassortment process may involve homologousrecombination or the natural property of quasi-repeated sequences todirect their own evolution.

[0107] Repeated or “quasi-repeated” sequences play a role in geneticinstability. In the present invention, “quasi-repeats” are repeats thatare not restricted to their original unit structure. Quasi-repeatedunits can be presented as an array of sequences in a construct;consecutive units of similar sequences. Once ligated, the junctionsbetween the consecutive sequences become essentially invisible and thequasi-repetitive nature of the resulting construct is now continuous atthe molecular level. The deletion process the cell performs to reducethe complexity of the resulting construct operates between thequasi-repeated sequences. The quasi-repeated units provide a practicallylimitless repertoire of templates upon which slippage events can occur.The constructs containing the quasi-repeats thus effectively providesufficient molecular elasticity that deletion (and potentiallyinsertion) events can occur virtually anywhere within thequasi-repetitive units.

[0108] When the quasi-repeated sequences are all ligated in the sameorientation, for instance head to tail or vice versa, the cell cannotdistinguish individual units. Consequently, the reductive process canoccur throughout the sequences. In contrast, when for example, the unitsare presented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, it is preferable with thepresent method that the sequences are in the same orientation. Randomorientation of quasi-repeated sequences will result in the loss ofreassortment efficiency, while consistent orientation of the sequenceswill offer the highest efficiency. However, while having fewer of thecontiguous sequences in the same orientation decreases the efficiency,it may still provide sufficient elasticity for the effective recovery ofnovel molecules. Constructs can be made with the quasi-repeatedsequences in the same orientation to allow higher efficiency.

[0109] Sequences can be assembled in a head to tail orientation usingany of a variety of methods, including the following:

[0110] a) Primers that include a poly-A head and poly-T tail which whenmade single-stranded would provide orientation can be utilized. This isaccomplished by having the first few bases of the primers made from RNAand hence easily removed RNAseH.

[0111] b) Primers that include unique restriction cleavage sites can beutilized. Multiple sites, a battery of unique sequences, and repeatedsynthesis and ligation steps would be required.

[0112] c) The inner few bases of the primer could be thiolated and anexonuclease used to produce properly tailed molecules.

[0113] The recovery of the re-assorted sequences relies on theidentification of cloning vectors with a reduced repetitive index (RI).The re-assorted encoding sequences can then be recovered byamplification. The products are re-cloned and expressed. The recovery ofcloning vectors with reduced RI can be affected by:

[0114] 1) The use of vectors only stably maintained when the constructis reduced in complexity.

[0115] 2) The physical recovery of shortened vectors by physicalprocedures. In this case, the cloning vector would be recovered usingstandard plasmid isolation procedures and size fractionated on either anagarose gel, or column with a low molecular weight cut off utilizingstandard procedures.

[0116] 3) The recovery of vectors containing interrupted genes which canbe selected when insert size decreases.

[0117] 4) The use of direct selection techniques with an expressionvector and the appropriate selection.

[0118] Encoding sequences (for example, genes) from related organismsmay demonstrate a high degree of homology and encode quite diverseprotein products. These types of sequences are particularly useful inthe present invention as quasi-repeats. However, while the examplesillustrated below demonstrate the reassortment of nearly identicaloriginal encoding sequences (quasi-repeats), this process is not limitedto such nearly identical repeats.

[0119] The following example demonstrates a method of the invention.Encoding nucleic acid sequences (quasi-repeats) derived from three (3)unique species are described. Each sequence encodes a protein with adistinct set of properties. Each of the sequences differs by a single ora few base pairs at a unique position in the sequence. Thequasi-repeated sequences are separately or collectively amplified andligated into random assemblies such that all possible permutations andcombinations are available in the population of ligated molecules. Thenumber of quasi-repeat units can be controlled by the assemblyconditions. The average number of quasi-repeated units in a construct isdefined as the repetitive index (RI).

[0120] Once formed, the constructs may, or may not be size fractionatedon an agarose gel according to published protocols, inserted into acloning vector, and transfected into an appropriate host cell. The cellsare then propagated and “reductive reassortment” is effected. The rateof the reductive reassortment process may be stimulated by theintroduction of DNA damage if desired. Whether the reduction in RI ismediated by deletion formation between repeated sequences by an“intra-molecular” mechanism, or mediated by recombination-like eventsthrough “inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations.

[0121] Optionally, the method comprises the additional step of screeningthe library members of the shuffled pool to identify individual shuffledlibrary members having the ability to bind or otherwise interact, orcatalyze a particular reaction (e.g., such as catalytic domain of anenzyme) with a predetermined macromolecule, such as for example aproteinaceous receptor, an oligosaccharide, viron, or otherpredetermined compound or structure.

[0122] The polypeptides that are identified from such libraries can beused for therapeutic, diagnostic, research and related purposes (e.g.,catalysts, solutes for increasing osmolarity of an aqueous solution, andthe like), and/or can be subjected to one or more additional cycles ofshuffling and/or selection.

[0123] In another aspect, it is envisioned that prior to or duringrecombination or reassortment, polynucleotides generated by the methodof the invention can be subjected to agents or processes which promotethe introduction of mutations into the original polynucleotides. Theintroduction of such mutations would increase the diversity of resultinghybrid polynucleotides and polypeptides encoded therefrom. The agents orprocesses which promote mutagenesis can include, but are not limitedto:(+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N3-Adenine(See Sun and Hurley, (1992); an N-acelylated or deacetylated4′-fluro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See, for example, van de Poll et al. (1992)); or a N-acetylated ordeacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See also, van de Poll et al. (1992), pp. 751-758); trivalent chromium,a trivalent chromium salt, a polycyclic aromatic hydrocarbon (PAH) DNAadduct capable of inhibiting DNA replication, such as7-bromomethyl-benz[α]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[α]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”), and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine (“N-hydroxy-PhIP”). Especiallypreferred means for slowing or halting PCR amplification consist of UVlight (+)-CC-1065 and (+)-CC-1065-(N3-Adenine). Particularly encompassedmeans are DNA adducts or polynucleotides comprising the DNA adducts fromthe polynucleotides or polynucleotides pool, which can be released orremoved by a process including heating the solution comprising thepolynucleotides prior to further processing.

[0124] In another aspect the invention is directed to a method ofproducing recombinant proteins having biological activity by treating asample comprising double-stranded template polynucleotides encoding awild-type protein under conditions according to the invention whichprovide for the production of hybrid or re-assorted polynucleotides.

[0125] The invention also provides for the use of proprietary codonprimers (containing a degenerate N,N,N sequence) to introduce pointmutations into a polynucleotide, so as to generate a set of progenypolypeptides in which a full range of single amino acid substitutions isrepresented at each amino acid position (gene site saturated mutagenesis(GSSM)). The oligos used are comprised contiguously of a firsthomologous sequence, a degenerate N,N,N sequence, and preferably but notnecessarily a second homologous sequence. The downstream progenytranslational products from the use of such oligos include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,N sequence includes codons for all 20amino acids.

[0126] In one aspect, one such degenerate oligo (comprised of onedegenerate N,N,N cassette) is used for subjecting each original codon ina parental polynucleotide template to a full range of codonsubstitutions. In another aspect, at least two degenerate N,N,Ncassettes are used—either in the same oligo or not, for subjecting atleast two original codons in a parental polynucleotide template to afull range of codon substitutions. Thus, more than one N,N,N sequencecan be contained in one oligo to introduce amino acid mutations at morethan one site. This plurality of N,N,N sequences can be directlycontiguous, or separated by one or more additional nucleotidesequence(s). In another aspect, oligos serviceable for introducingadditions and deletions can be used either alone or in combination withthe codons containing an N,N,N sequence, to introduce any combination orpermutation of amino acid additions, deletions, and/or substitutions.

[0127] In a particular exemplification, it is possible to simultaneouslymutagenize two or more contiguous amino acid positions using an oligothat contains contiguous N,N,N triplets, ie. a degenerate (NNN)_(n)sequence.

[0128] In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,N sequence. Forexample, it may be desirable in some instances to use (e.g. in an oligo)a degenerate triplet sequence comprised of only one N, where said N canbe in the first second or third position of the triplet. Any other basesincluding any combinations and permutations thereof can be used in theremaining two positions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g., in an oligo) a degenerateN,N,N triplet sequence, N,N,G/T, or an N,N, G/C triplet sequence.

[0129] It is appreciated, however, that the use of a degenerate triplet(such as N,N,G/T or an N,N, G/C triplet sequence) as disclosed in theinstant invention is advantageous for several reasons. In one aspect,this invention provides a means to systematically and fairly easilygenerate the substitution of the full range of possible amino acids (fora total of 20 amino acids) into each and every amino acid position in apolypeptide. Thus, for a 100 amino acid polypeptide, the inventionprovides a way to systematically and fairly easily generate 2000distinct species (i.e., 20 possible amino acids per position times 100amino acid positions). It is appreciated that there is provided, throughthe use of an oligo containing a degenerate N,N,G/T or an N,N, G/Ctriplet sequence, 32 individual sequences that code for 20 possibleamino acids. Thus, in a reaction vessel in which a parentalpolynucleotide sequence is subjected to saturation mutagenesis using onesuch oligo, there are generated 32 distinct progeny polynucleotidesencoding 20 distinct polypeptides. In contrast, the use of anon-degenerate oligo in site-directed mutagenesis leads to only oneprogeny polypeptide product per reaction vessel.

[0130] This invention also provides for the use of nondegenerate oligos,which can optionally be used in combination with degenerate primersdisclosed. It is appreciated that in some situations, it is advantageousto use nondegenerate oligos to generate specific point mutations in aworking polynucleotide. This provides a means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

[0131] Thus, in a preferred embodiment of this invention, eachsaturation mutagenesis reaction vessel contains polynucleotides encodingat least 20 progeny polypeptide molecules such that all 20 amino acidsare represented at the one specific amino acid position corresponding tothe codon position mutagenized in the parental polynucleotide. The32-fold degenerate progeny polypeptides generated from each saturationmutagenesis reaction vessel can be subjected to clonal amplification(e.g., cloned into a suitable E. coli host using an expression vector)and subjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide), it can besequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

[0132] It is appreciated that upon mutagenizing each and every aminoacid position in a parental polypeptide using saturation mutagenesis asdisclosed herein, favorable amino acid changes may be identified at morethan one amino acid position. One or more new progeny molecules can begenerated that contain a combination of all or part of these favorableamino acid substitutions. For example, if 2 specific favorable aminoacid changes are identified in each of 3 amino acid positions in apolypeptide, the permutations include 3 possibilities at each position(no change from the original amino acid, and each of two favorablechanges) and 3 positions. Thus, there are 3×3×3 or 27 totalpossibilities, including 7 that were previously examined −6 single pointmutations (i.e., 2 at each of three positions) and no change at anyposition.

[0133] In yet another aspect, site-saturation mutagenesis can be usedtogether with shuffling, chimerization, recombination and othermutagenizing processes, along with screening. This invention providesfor the use of any mutagenizing process(es), including saturationmutagenesis, in an iterative manner. In one exemplification, theiterative use of any mutagenizing process(es) is used in combinationwith screening.

[0134] Thus, in a non-limiting exemplification, this invention providesfor the use of saturation mutagenesis in combination with additionalmutagenization processes, such as process where two or more relatedpolynucleotides are introduced into a suitable host cell such that ahybrid polynucleotide is generated by recombination and reductivereassortment.

[0135] In addition to performing mutagenesis along the entire sequenceof a gene, the instant invention provides that mutagenesis can be use toreplace each of any number of bases in a polynucleotide sequence,wherein the number of bases to be mutagenized is preferably everyinteger from 15 to 100,000. Thus, instead of mutagenizing every positionalong a molecule, one can subject every or a discrete number of bases(preferably a subset totaling from 15 to 100,000) to mutagenesis.Preferably, a separate nucleotide is used for mutagenizing each positionor group of positions along a polynucleotide sequence. A group of 3positions to be mutagenized may be a codon. The mutations are preferablyintroduced using a mutagenic primer, containing a heterologous cassette,also referred to as a mutagenic cassette. Preferred cassettes can havefrom 1 to 500 bases. Each nucleotide position in such heterologouscassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T, C/G/T, A/G/T,A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E canbe referred to as a designer oligo).

[0136] In a general sense, saturation mutagenesis is comprised ofmutagenizing a complete set of mutagenic cassettes (wherein eachcassette is preferably about 1-500 bases in length) in definedpolynucleotide sequence to be mutagenized (wherein the sequence to bemutagenized is preferably from about 15 to 100,000 bases in length).Thus, a group of mutations (ranging from 1 to 100 mutations) isintroduced into each cassette to be mutagenized. A grouping of mutationsto be introduced into one cassette can be different or the same from asecond grouping of mutations to be introduced into a second cassetteduring the application of one round of saturation mutagenesis. Suchgroupings are exemplified by deletions, additions, groupings ofparticular codons, and groupings of particular nucleotide cassettes.

[0137] Defined sequences to be mutagenized include a whole gene,pathway, cDNA, an entire open reading frame (ORF), and entire promoter,enhancer, repressorltransactivator, origin of replication, intron,operator, or any polynucleotide functional group. Generally, a “definedsequences” for this purpose may be any polynucleotide that a 15base-polynucleotide sequence, and polynucleotide sequences of lengthsbetween 15 bases and 15,000 bases (this invention specifically namesevery integer in between). Considerations in choosing groupings ofcodons include types of amino acids encoded by a degenerate mutageniccassette.

[0138] In a particularly preferred exemplification a grouping ofmutations that can be introduced into a mutagenic cassette, thisinvention specifically provides for degenerate codon substitutions(using degenerate oligos) that code for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids at each position, anda library of polypeptides encoded thereby.

[0139] One aspect of the invention is an isolated nucleic acidcomprising one of the sequences of SEQ ID NO.:3, and sequencessubstantially identical thereto, the sequences complementary thereto, ora fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,150, 200, 300, 400, or 500 consecutive bases of SEQ ID NO.:3 (or thesequence complementary thereto). The isolated, nucleic acids maycomprise DNA, including cDNA, genomic DNA, and synthetic DNA. The DNAmay be double-stranded or single-stranded, and if single stranded may bethe coding strand or non-coding (anti-sense) strand. Alternatively, theisolated nucleic acids may comprise RNA.

[0140] As discussed in more detail below, the isolated nucleic acids ofSEQ ID NO.:3, and sequences substantially identical thereto, may be usedto prepare the polypeptides of SEQ ID NO.:4, and sequences substantiallyidentical thereto, or fragments comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids of SEQ ID NO.:4,and sequences substantially identical thereto.

[0141] Accordingly, another aspect of the invention is an isolatednucleic acid which encodes SEQ ID NO.:4, and sequences substantiallyidentical thereto, or fragments comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids of SEQ ID NO.:4.The coding sequences of these nucleic acids may be identical to thecoding sequence of SEQ ID NO.:3, or a fragment thereof or may bedifferent coding sequences which encode SEQ ID NO.:4, sequencessubstantially identical thereto, and fragments having at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids ofSEQ ID NO.:4, as a result of the redundancy or degeneracy of the geneticcode. The genetic code is well known to those of skill in the art andcan be obtained, for example, on page 214 of B. Lewin, Genes VI, OxfordUniversity Press, 1997, the disclosure of which is incorporated hereinby reference.

[0142] The isolated nucleic acid which encodes SEQ ID NO.:4, andsequences substantially identical thereto, may include, but is notlimited to:only the coding sequence SEQ ID NO.:3, and sequencessubstantially identical thereto, and additional coding sequences, suchas leader sequences or proprotein sequences and non-coding sequences,such as introns or non-coding sequences 5′ and/or 3′ of the codingsequence. Thus, as used herein, the term “polynucleotide encoding apolypeptide” encompasses a polynucleotide which includes only the codingsequence for the polypeptide as well as a polynucleotide which includesadditional coding and/or non-coding sequence.

[0143] Alternatively, the nucleic acid sequence of SEQ ID NO.:3, andsequences substantially identical thereto, may be mutagenized usingconventional techniques, such as site directed mutagenesis, or othertechniques familiar to those skilled in the art, to introduce silentchanges into the polynucleotides of SEQ ID NO.: 3, and sequencessubstantially identical thereto. As used herein, “silent changes”include, for example, changes which do not alter the amino acid sequenceencoded by the polynucleotide. Such changes may be desirable in order toincrease the level of the polypeptide produced by host cells containinga vector encoding the polypeptide by introducing codons or codon pairswhich occur frequently in the host organism.

[0144] The invention also relates to polynucleotides which havenucleotide changes which result in amino acid substitutions, additions,deletions, fusions and truncations in SEQ ID NO.:4, and sequencessubstantially identical thereto. Such nucleotide changes may beintroduced using techniques such as site directed mutagenesis, randomchemical mutagenesis, exonuclease III deletion, and other recombinantDNA techniques. Alternatively, such nucleotide changes may be naturallyoccurring allelic variants which are isolated by identifying nucleicacids which specifically hybridize to probes comprising at least 10, 15,20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutivebases of SEQ ID NO.:3, and sequences substantially identical thereto (orthe sequences complementary thereto) under conditions of high, moderate,or low stringency as provided herein.

[0145] The isolated nucleic acids of SEQ ID NO.:3, and sequencessubstantially identical thereto, the sequences complementary thereto, ora fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,150, 200, 300, 400, or 500 consecutive bases of SEQ ID NO.:3, andsequences substantially identical thereto, or the sequencescomplementary thereto may also be used as probes to determine whether abiological sample, such as a soil sample, contains an organism having anucleic acid sequence of the invention or an organism from which thenucleic acid was obtained. In such procedures, a biological samplepotentially harboring the organism from which the nucleic acid wasisolated is obtained and nucleic acids are obtained from the sample. Thenucleic acids are contacted with the probe under conditions which permitthe probe to specifically hybridize to any complementary sequences fromwhich are present therein.

[0146] Where necessary, conditions which permit the probe tospecifically hybridize to complementary sequences may be determined byplacing the probe in contact with complementary sequences from samplesknown to contain the complementary sequence as well as control sequenceswhich do not contain the complementary sequence. Hybridizationconditions, such as the salt concentration of the hybridization buffer,the formamide concentration of the hybridization buffer, or thehybridization temperature, may be varied to identify conditions whichallow the probe to hybridize specifically to complementary nucleicacids.

[0147] If the sample contains the organism from which the nucleic acidwas isolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product.

[0148] Many methods for using the labeled probes to detect the presenceof complementary nucleic acids in a sample are familiar to those skilledin the art. These include Southern Blots, Northern Blots, colonyhybridization procedures, and dot blots. Protocols for each of theseprocedures are provided in Ausubel et al. Current Protocols in MolecularBiology, John Wiley 503 Sons, Inc. (1997) and Sambrook et al., MolecularCloning:A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press(1989), the entire disclosures of which are incorporated herein byreference.

[0149] Alternatively, more than one probe (at least one of which iscapable of specifically hybridizing to any complementary sequences whichare present in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). Typically, the probes compriseoligonucleotides. In one embodiment, the amplification reaction maycomprise a PCR reaction. PCR protocols are described in Ausubel andSambrook, supra. Alternatively, the amplification may comprise a ligasechain reaction, 3SR, or strand displacement reaction. (See Barany, F.,“The Ligase Chain Reaction in a PCR World”, PCR Methods and Applications1:5-16, 1991; E. Fahy et al., “Self-sustained Sequence Replication (3SR):An Isothermal Transcription-based Amplification System Alternativeto PCR”, PCR Methods and Applications 1:25-33, 1991; and Walker G. T. etal., “Strand Displacement Amplification-an Isothermal in vitro DNAAmplification Technique”, Nucleic Acid Research 20:1691-1696, 1992, thedisclosures of which are incorporated herein by reference in theirentireties). In such procedures, the nucleic acids in the sample arecontacted with the probes, the amplification reaction is performed, andany resulting amplification product is detected. The amplificationproduct may be detected by performing gel electrophoresis on thereaction products and staining the gel with an interculator such asethidium bromide. Alternatively, one or more of the probes may belabeled with a radioactive isotope and the presence of a radioactiveamplification product may be detected by autoradiography after gelelectrophoresis.

[0150] Probes derived from sequences near the ends of SEQ ID NO.:3, andsequences substantially identical thereto, may also be used inchromosome walking procedures to identify clones containing genomicsequences located adjacent to SEQ ID NO.:3, and sequences substantiallyidentical thereto. Such methods allow the isolation of genes whichencode additional proteins from the host organism.

[0151] The isolated nucleic acids of SEQ ID NO.:3, and sequencessubstantially identical thereto, the sequences complementary thereto, ora fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,150, 200, 300, 400, or 500 consecutive bases of SEQ ID NO.:3, andsequences substantially identical thereto, or the sequencescomplementary thereto may be used as probes to identify and isolaterelated nucleic acids. In some embodiments, the related nucleic acidsmay be cDNAs or genomic DNAs from organisms other than the one fromwhich the nucleic acid was isolated. For example, the other organismsmay be related organisms. In such procedures, a nucleic acid sample iscontacted with the probe under conditions which permit the probe tospecifically hybridize to related sequences. Hybridization of the probeto nucleic acids from the related organism is then detected using any ofthe methods described above.

[0152] In nucleic acid hybridization reactions, the conditions used toachieve a particular level of stringency will vary, depending on thenature of the nucleic acids being hybridized. For example, the length,degree of complementarity, nucleotide sequence composition (e.g., GC v.AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

[0153] Hybridization may be carried out under conditions of lowstringency, moderate stringency or high stringency. As an example ofnucleic acid hybridization, a polymer membrane containing immobilizeddenatured nucleic acids is first prehybridized for 30 minutes at 45° C.in a solution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10X Denhardt's, and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4−9×10⁸ cpm/ug) of ³²pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature in 1× SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh1× SET at T_(m)-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

[0154] By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, T_(m), isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the T_(m) for a particular probe. The melting temperature of theprobe may be calculated using the following formulas:

[0155] For probes between 14 and 70 nucleotides in length the meltingtemperature (T_(m)) is calculated using the formula:T_(m)=81.5+16.6(log[Na+])+0.41(fraction G+C)-(600/N) where N is the length of the probe.

[0156] If the hybridization is carried out in a solution containingformamide, the melting temperature may be calculated using theequation:T_(m)=81.5+16.6(log [Na+])+0.41(fraction G+C)-(0.63%formamide)-(600/N) where N is the length of the probe.

[0157] Prehybridization may be carried out in 6× SSC, 5× Denhardt'sreagent, 0.5% SDS, 100μg denatured fragmented salmon sperm DNA or 6×SSC, 5× Denhardt's reagent, 0.5% SDS, 100 μg denatured fragmented salmonsperm DNA, 50% formamide. The formulas for SSC and Denhardt's solutionsare listed in Sambrook et al., supra.

[0158] Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the T_(m). Forshorter probes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the T_(m). Typically, for hybridizations in6× SSC, the hybridization is conducted at approximately 68° C. Usually,for hybridizations in 50% formamide containing solutions, thehybridization is conducted at approximately 42° C.

[0159] All of the foregoing hybridizations would be considered to beunder conditions of high stringency.

[0160] Following hybridization, the filter is washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content), and the nucleic acid type (e.g. RNA v. DNA). Examples ofprogressively higher stringency condition washes are as follows:2× SSC,0.1% SDS at room temperature for 15 minutes (low stringency); 0.1× SSC,0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1× SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1× SSC at room temperature. The examplesabove are merely illustrative of one set of conditions that can be usedto wash filters. One of skill in the art would know that there arenumerous recipes for different stringency washes. Some other examplesare given below.

[0161] Nucleic acids which have hybridized to the probe are identifiedby autoradiography or other conventional techniques.

[0162] The above procedure may be modified to identify nucleic acidshaving decreasing levels of homology to the probe sequence. For example,to obtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na+ concentration of approximately1M. Following hybridization, the filter may be washed with 2× SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate” conditions above 50° C. and “low” conditions below 50°C. A specific example of“moderate” hybridization conditions is when theabove hybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

[0163] Alternatively, the hybridization may be carried out in buffers,such as 6× SSC, containing formamide at a temperature of 42° C. In thiscase, the concentration of formamide in the hybridization buffer may bereduced in 5% increments from 50% to 0% to identify clones havingdecreasing levels of homology to the probe. Following hybridization, thefilter may be washed with 6× SSC, 0.5% SDS at 50° C. These conditionsare considered to be “moderate” conditions above 25% formamide and “low”conditions below 25% formamide. A specific example of “moderate”hybridization conditions is when the above hybridization is conducted at30% formamide. A specific example of “low stringency” hybridizationconditions is when the above hybridization is conducted at 10%formamide.

[0164] For example, the preceding methods may be used to isolate nucleicacids having a sequence with at least about 97%, at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least65%, at least 60%, at least 55%, or at least 50% homology to SEQ IDNO.:3, and sequences substantially identical thereto, or fragmentscomprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150,200, 300, 400, or 500 consecutive bases thereof, and the sequencescomplementary thereto. Homology may be measured using the alignmentalgorithm. For example, the homologous polynucleotides may have a codingsequence which is a naturally occurring allelic variant of one of thecoding sequences described herein. Such allelic variants may have asubstitution, deletion or addition of one or more nucleotides whencompared to the nucleic acids of SEQ ID NO.:3 or the sequencescomplementary thereto.

[0165] Additionally, the above procedures may be used to isolate nucleicacids which encode polypeptides having at least about 99%, 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least65%, at least 60%, at least 55%, or at least 50% homology to apolypeptide having the sequence of SEQ ID NO.: 4, and sequencessubstantially identical thereto, or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof as determined using a sequence alignment algorithm (e.g., suchas the FASTA version 3.0t78 algorithm with the default parameters).

[0166] Another aspect of the invention is an isolated or purifiedpolypeptide comprising the sequence of SEQ ID NO.:3, and sequencessubstantially identical thereto, or fragments comprising at least about5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive aminoacids thereof. As discussed above, such polypeptides may be obtained byinserting a nucleic acid encoding the polypeptide into a vector suchthat the coding sequence is operably linked to a sequence capable ofdriving the expression of the encoded polypeptide in a suitable hostcell. For example, the expression vector may comprise a promoter, aribosome binding site for translation initiation and a transcriptionterminator. The vector may also include appropriate sequences foramplifying expression.

[0167] Promoters suitable for expressing the polypeptide or fragmentthereof in bacteria include the E. coli lac or trp promoters, the lacpromoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gptpromoter, the lambda P_(R) promoter, the lambda P_(L) promoter,promoters from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), and the acid phosphatase promoter.Fungal promoters include the ∀ factor promoter. Eukaryotic promotersinclude the CMV immediate early promoter, the HSV thymidine kinasepromoter, heat shock promoters, the early and late SV40 promoter, LTRsfrom retroviruses, and the mouse metallothionein-I promoter. Otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses may also be used.

[0168] Mammalian expression vectors may also comprise an origin ofreplication, any necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, transcriptional terminationsequences, and 5′ flanking nontranscribed sequences. In someembodiments, DNA sequences derived from the SV40 splice andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

[0169] Vectors for expressing the polypeptide or fragment thereof ineukaryotic cells may also contain enhancers to increase expressionlevels. Enhancers are cis-acting elements of DNA, usually from about 10to about 300 bp in length that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and the adenovirus enhancers.

[0170] In addition, the expression vectors typically contain one or moreselectable marker genes to permit selection of host cells containing thevector. Such selectable markers include genes encoding dihydrofolatereductase or genes conferring neomycin resistance for eukaryotic cellculture, genes conferring tetracycline or ampicillin resistance in E.coli, and the S. cerevisiae TRP1 gene.

[0171] In some embodiments, the nucleic acid encoding SEQ ID NO.:4, andsequences substantially identical thereto, or fragments comprising atleast about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof is assembled in appropriate phase with aleader sequence capable of directing secretion of the translatedpolypeptide or fragment thereof. Optionally, the nucleic acid can encodea fusion polypeptide in which SEQ ID NO.:4, and sequences substantiallyidentical thereto, or fragments comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof is fusedto heterologous peptides or polypeptides, such as N-terminalidentification peptides which impart desired characteristics, such asincreased stability or simplified purification.

[0172] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is ligated to thedesired position in the vector following digestion of the insert and thevector with appropriate restriction endonucleases. Alternatively, bluntends in both the insert and the vector may be ligated. A variety ofcloning techniques are disclosed in Ausubel et al. Current Protocols inMolecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et al.,Molecular Cloning:A Laboratory Manual 2nd Ed., Cold Spring HarborLaboratory Press (1989), the entire disclosures of which areincorporated herein by reference. Such procedures and others are deemedto be within the scope of those skilled in the art.

[0173] The vector may be, for example, in the form of a plasmid, a viralparticle, or a phage. Other vectors include chromosomal, nonchromosomaland synthetic DNA sequences, derivatives of SV40; bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, and pseudorabies. A variety of cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning:A Laboratory Manual 2ndEd., Cold Spring Harbor, N.Y., (1989), the disclosure of which is herebyincorporated by reference.

[0174] Particular bacterial vectors which may be used include thecommercially available plasmids comprising genetic elements of the wellknown cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, Wis., USA)pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174 pBluescript II KS, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia), pKK232-8 and pCM7. Particular eukaryoticvectors include pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV,pMSG, and pSVL (Pharmacia). However, any other vector may be used aslong as it is replicable and viable in the host cell.

[0175] The host cell may be any of the host cells familiar to thoseskilled in the art, including prokaryotic cells, eukaryotic cells,mammalian cells, insect cells, or plant cells. As representativeexamples of appropriate hosts, there may be mentioned: bacterial cells,such as E. coli, Streptomyces, Bacillus subtilis Salmonella typhimuriumand various species within the genera Pseudomonas, Streptomyces, andStaphylococcus, fungal cells, such as yeast, insect cells such asDrosophila S2 and Spodoptera Sf9, animal cells such as CHO, COS or Bowesmelanoma, and adenoviruses. The selection of an appropriate host iswithin the abilities of those skilled in the art.

[0176] The vector may be introduced into the host cells using any of avariety of techniques, including transformation, transfection,transduction, viral infection, gene guns, or Ti-mediated gene transfer.Particular methods include calcium phosphate transfection, DEAE-Dextranmediated transfection, lipofection, or electroporation (Davis, L.,Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

[0177] Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

[0178] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract is retainedfor further purification. Microbial cells employed for expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents. Such methods are well known to those skilled in the art.The expressed polypeptide or fragment thereof can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the polypeptide. Ifdesired, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

[0179] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts (described byGluzrnan, Cell, 23:175, 1981), and other cell lines capable ofexpressing proteins from a compatible vector, such as the C127, 3T3,CHO, HeLa and BHK cell lines.

[0180] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Depending upon the host employed in a recombinant production procedure,the polypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

[0181] Alternatively, SEQ ID NO.:4, and sequences substantiallyidentical thereto, or fragments comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof can besynthetically produced by conventional peptide synthesizers. In otherembodiments, fragments or portions of the polypeptides may be employedfor producing the corresponding full-length polypeptide by peptidesynthesis; therefore, the fragments may be employed as intermediates forproducing the full-length polypeptides.

[0182] Cell-free translation systems can also be employed to produce SEQID NO.:4, and sequences substantially identical thereto, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof using mRNAs transcribed from a DNAconstruct comprising a promoter operably linked to a nucleic acidencoding the polypeptide or fragment thereof. In some embodiments, theDNA construct may be linearized prior to conducting an in vitrotranscription reaction. The transcribed mRNA is then incubated with anappropriate cell-free translation extract, such as a rabbit reticulocyteextract, to produce the desired polypeptide or fragment thereof.

[0183] The invention also relates to variants of SEQ ID NO.:4, andsequences substantially identical thereto, or fragments comprising atleast 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutiveamino acids thereof. The term “variant” includes derivatives or analogsof these polypeptides. In particular, the variants may differ in aminoacid sequence from SEQ ID NO.:4, and sequences substantially identicalthereto, by one or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

[0184] The variants may be naturally occurring or created in vitro. Inparticular, such variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures.

[0185] Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. Typically, thesenucleotide differences result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

[0186] For example, variants may be created using error prone PCR. Inerror prone PCR, PCR is performed under conditions where the copyingfidelity of the DNA polymerase is low, such that a high rate of pointmutations is obtained along the entire length of the PCR product. Errorprone PCR is described in Leung, D. W., et al., Technique, I 11- 15,1989) and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33,1992, the disclosure of which is incorporated herein by reference in itsentirety. Briefly, in such procedures, nucleic acids to be mutagenizedare mixed with PCR primers, reaction buffer, MgCl₂, MnCl₂, Taqpolymerase and an appropriate concentration of dNTPs for achieving ahigh rate of point mutation along the entire length of the PCR product.For example, the reaction may be performed using 20 finoles of nucleicacid to be mutagenized, 30 pmole of each PCR primer, a reaction buffercomprising 50 mM KCl, 10mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mMMgCl₂, 0.5 mM MnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mMdATP, 1 mM dCTP, and 1 mM dTTP. PCR may be performed for 30 cycles of94° C. for 1 min, 45° C. for 1 min, and 72° C. for 1 min. However, itwill be appreciated that these parameters may be varied as appropriate.The mutagenized nucleic acids are cloned into an appropriate vector andthe activities of the polypeptides encoded by the mutagenized nucleicacids is evaluated.

[0187] Variants may also be created using oligonucleotide directedmutagenesis to generate site-specific mutations in any cloned DNA ofinterest. Oligonucleotide mutagenesis is described in Reidhaar-Olson, J.F. & Sauer, R. T., et al., Science, 241:53-57, 1988, the disclosure ofwhich is incorporated herein by reference in its entirety. Briefly, insuch procedures a plurality of double stranded oligonucleotides bearingone or more mutations to be introduced into the cloned DNA aresynthesized and inserted into the cloned DNA to be mutagenized. Clonescontaining the mutagenized DNA are recovered and the activities of thepolypeptides they encode are assessed.

[0188] Another method for generating variants is assembly PCR. AssemblyPCR involves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in U.S. Pat. No.5,965,408, filed Jul. 9, 1996, entitled, “Method of DNA Reassembly byInterrupting Synthesis”, the disclosure of which is incorporated hereinby reference in its entirety.

[0189] Still another method of generating variants is sexual PCRmutagenesis. In sexual PCR mutagenesis, forced homologous recombinationoccurs between DNA molecules of different but highly related DNAsequence in vitro, as a result of random fragmentation of the DNAmolecule based on sequence homology, followed by fixation of thecrossover by primer extension in a PCR reaction. Sexual PCR mutagenesisis described in Stemmer, W. P., PNAS, USA, 91:10747-10751, 1994, thedisclosure of which is incorporated herein by reference. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNAse to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/:1 in a solution of 0.2 mM of each dNTP, 2.2mM MgCl2, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime:94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some embodiments,oligonucleotides may be included in the PCR reactions. In otherembodiments, the Klenow fragment of DNA polymerase I may be used in afirst set of PCR reactions and Taq polymerase may be used in asubsequent set of PCR reactions. Recombinant sequences are isolated andthe activities of the polypeptides they encode are assessed.

[0190] Variants may also be created by in vivo mutagenesis. In someembodiments, random mutations in a sequence of interest are generated bypropagating the sequence of interest in a bacterial strain, such as anE. coli strain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described in PCTPublication No. WO 91/16427, published Oct. 31, 1991, entitled “Methodsfor Phenotype Creation from Multiple Gene Populations” the disclosure ofwhich is incorporated herein by reference in its entirety.

[0191] Variants may also be generated using cassette mutagenesis. Incassette mutagenesis a small region of a double stranded DNA molecule isreplaced with a synthetic oligonucleotide “cassette” that differs fromthe native sequence. The oligonucleotide often contains completelyand/or partially randomized native sequence.

[0192] Recursive ensemble mutagenesis may also be used to generatevariants. Recursive ensemble mutagenesis is an algorithm for proteinengineering (protein mutagenesis) developed to produce diversepopulations of phenotypically related mutants whose members differ inamino acid sequence. This method uses a feedback mechanism to controlsuccessive rounds of combinatorial cassette mutagenesis. Recursiveensemble mutagenesis is described in Arkin, A. P. and Youvan, D.C.,PNAS, USA, 89:7811-7815, 1992, the disclosure of which is incorporatedherein by reference in its entirety.

[0193] In some embodiments, variants are created using exponentialensemble mutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described inDelegrave, S. and Youvan, D. C., Biotechnology Research, 11:1548-1552,1993, the disclosure of which incorporated herein by reference in itsentirety. Random and site-directed mutagenesis are described in Arnold,F. H., Current Opinion in Biotechnology, 4:450-455, 1993, the disclosureof which is incorporated herein by reference in its entirety.

[0194] In some embodiments, the variants are created using shufflingprocedures wherein portions of a plurality of nucleic acids which encodedistinct polypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in U.S. Pat.No. 5,965,408, filed July 9, 1996, entitled, “Method of DNA Reassemblyby Interrupting Synthesis”, and U.S. Pat. No. 5,939,250, filed May 22,1996, entitled, “Production of Enzymes Having Desired Activities byMutagenesis”, both of which are incorporated herein by reference.

[0195] The variants of SEQ ID NO.:4 may be variants in which one or moreof the amino acid residues of SEQ ID NO.:4 are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code.

[0196] Conservative substitutions are those that substitute a givenamino acid in a polypeptide by another amino acid of likecharacteristics. Typically seen as conservative substitutions are thefollowing replacements:replacements of an aliphatic amino acid such asAlanine, Valine, Leucine and Isoleucine with another aliphatic aminoacid; replacement of a Serine with a Threonine or vice versa;replacement of an acidic residue such as Aspartic acid and Glutamic acidwith another acidic residue; replacement of a residue bearing an amidegroup, such as Asparagine and Glutamine, with another residue bearing anamide group; exchange of a basic residue such as Lysine and Argininewith another basic residue; and replacement of an aromatic residue suchas Phenylalanine, Tyrosine with another aromatic residue.

[0197] Other variants are those in which one or more of the amino acidresidues of SEQ ID NO.:4 includes a substituent group.

[0198] Still other variants are those in which the polypeptide isassociated with another compound, such as a compound to increase thehalf-life of the polypeptide (for example, polyethylene glycol).

[0199] Additional variants are those in which additional amino acids arefused to the polypeptide, such as a leader sequence, a secretorysequence, a proprotein sequence or a sequence which facilitatespurification, enrichment, or stabilization of the polypeptide.

[0200] In some embodiments, the fragments, derivatives and analogsretain the same biological function or activity as SEQ ID NO.:4, andsequences substantially identical thereto. In other embodiments, thefragment, derivative, or analog includes a proprotein, such that thefragment, derivative, or analog can be activated by cleavage of theproprotein portion to produce an active polypeptide.

[0201] Another aspect of the invention is polypeptides or fragmentsthereof which have at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or more thanabout 95% homology to SEQ ID NO.:4, and sequences substantiallyidentical thereto, or a fragment comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.Homology may be determined using any of the programs described abovewhich aligns the polypeptides or fragments being compared and determinesthe extent of amino acid identity or similarity between them. It will beappreciated that amino acid “homology” includes conservative amino acidsubstitutions such as those described above.

[0202] The polypeptides or fragments having homology to SEQ ID NO.:4,and sequences substantially identical thereto, or a fragment comprisingat least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof may be obtained by isolating the nucleicacids encoding them using the techniques described above.

[0203] Alternatively, the homologous polypeptides or fragments may beobtained through biochemical enrichment or purification procedures. Thesequence of potentially homologous polypeptides or fragments may bedetermined by proteolytic digestion, gel electrophoresis and/ormicrosequencing. The sequence of the prospective homologous polypeptideor fragment can be compared to SEQ ID NO.:4, and sequences substantiallyidentical thereto, or a fragment comprising at least about 5, 10, 15,20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereofusing any of the programs described above.

[0204] Another aspect of the invention is an assay for identifyingfragments or variants of SEQ ID NO.:4, and sequences substantiallyidentical thereto, which retain the enzymatic function of SEQ ID NO.:4,and sequences substantially identical thereto. For example the fragmentsor variants of said polypeptides, may be used to catalyze biochemicalreactions, which indicate that the fragment or variant retains theenzymatic activity of SEQ ID NO.:4.

[0205] The assay for determining if fragments of variants retain theenzymatic activity of SEQ ID NO.:4, and sequences substantiallyidentical thereto includes the steps of:contacting the polypeptidefragment or variant with a substrate molecule under conditions whichallow the polypeptide fragment or variant to function, and detectingeither a decrease in the level of substrate or an increase in the levelof the specific reaction product of the reaction between the polypeptideand substrate.

[0206] The polypeptide of SEQ ID NO.:4, and sequences substantiallyidentical thereto or fragments comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof may beused in a variety of applications. For example, the polypeptides orfragments thereof may be used to catalyze biochemical reactions. Inaccordance with one aspect of the invention, there is provided a processfor utilizing SEQ ID NO.:4, and sequences substantially identicalthereto or polynucleotides encoding such polypeptides for hydrolyzingglycosidic linkages. In such procedures, a substance containing aglycosidic linkage (e.g., a starch) is contacted with SEQ ID NO.:4, orsequences substantially identical thereto under conditions whichfacilitate the hydrolysis of the glycosidic linkage.

[0207] The polypeptide of SEQ ID NO.:4, and sequences substantiallyidentical thereto or fragments comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof, mayalso be used to generate antibodies which bind specifically to thepolypeptides or fragments. The resulting antibodies may be used inimmunoaffinity chromatography procedures to isolate or purify thepolypeptide or to determine whether the polypeptide is present in abiological sample. In such procedures, a protein preparation, such as anextract, or a biological sample is contacted with an antibody capable ofspecifically binding to SEQ ID NO.:4, and sequences substantiallyidentical thereto, or fragments comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.

[0208] In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to SEQ ID NO.:4, and sequences substantiallyidentical thereto, or fragment thereof. After a wash to removenon-specifically bound proteins, the specifically bound polypeptides areeluted.

[0209] The ability of proteins in a biological sample to bind to theantibody may be determined using any of a variety of procedures familiarto those skilled in the art. For example, binding may be determined bylabeling the antibody with a detectable label such as a fluorescentagent, an enzymatic label, or a radioisotope. Alternatively, binding ofthe antibody to the sample may be detected using a secondary antibodyhaving such a detectable label thereon. Particular assays include ELISAassays, sandwich assays, radioimmunoassays, and Western Blots.

[0210] Polyclonal antibodies generated against SEQ ID NO.:4, andsequences substantially identical thereto, or fragments comprising atleast 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutiveamino acids thereof can be obtained by direct injection of thepolypeptides into an animal or by administering the polypeptides to ananimal, for example, a nonhuman. The antibody so obtained will then bindthe polypeptide itself. In this manner, even a sequence encoding only afragment of the polypeptide can be used to generate antibodies which maybind to the whole native polypeptide. Such antibodies can then be usedto isolate the polypeptide from cells expressing that polypeptide.

[0211] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,Nature, 256:495-497, 1975, the disclosure of which is incorporatedherein by reference), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., Immunology Today 4:72, 1983, the disclosure ofwhich is incorporated herein by reference), and the EBV-hybridomatechnique (Cole, et al., 1985, in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96, the disclosure of which isincorporated herein by reference).

[0212] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778, the disclosure of which isincorporated herein by reference) can be adapted to produce single chainantibodies to SEQ ID NO.:4, and sequences substantially identicalthereto, or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40,50, 75, 100, or 150 consecutive amino acids thereof. Alternatively,transgenic mice may be used to express humanized antibodies to thesepolypeptides or fragments thereof.

[0213] Antibodies generated against SEQ ID NO.:4, and sequencessubstantially identical thereto, or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof may be used in screening for similar polypeptides from otherorganisms and samples. In such techniques, polypeptides from theorganism are contacted with the antibody and those polypeptides whichspecifically bind the antibody are detected. Any of the proceduresdescribed above may be used to detect antibody binding. One suchscreening assay is described in “Methods for Measuring CellulaseActivities”, Methods in Enzymology, Vol 160, pp. 87-116, which is herebyincorporated by reference in its entirety.

[0214] As used herein the term “nucleic acid sequence as set forth SEQID NO.:3” encompasses the nucleotide sequence SEQ ID NO.:3, andsequences substantially identical thereto, as well as sequenceshomologous to SEQ ID NO.:3, and fragments thereof and sequencescomplementary to all of the preceding sequences. The fragments includeportions of SEQ ID NO.:3, comprising at least 10, 15, 20, 25, 30, 35,40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive nucleotides ofSEQ ID NO.:3, and sequences substantially identical thereto. Homologoussequences and fragments of SEQ ID NO.:3, and sequences substantiallyidentical thereto, refer to a sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% homology tothese sequences. Homology may be determined using any of the computerprograms and parameters described herein, including FASTA version 3.0t78with the default parameters. Homologous sequences also include RNAsequences in which uridines replace the thymines in the nucleic acidsequences as set forth in SEQ ID NO.:3. The homologous sequences may beobtained using any of the procedures described herein or may result fromthe correction of a sequencing error. It will be appreciated that thenucleic acid sequence as set forth in SEQ ID NO.:3, and sequencessubstantially identical thereto, can be represented in the traditionalsingle character format (See the inside back cover of Stryer, Lubert.Biochemistry 3rd Ed., W. H Freeman & Co., New York.) or in any otherformat which records the identity of the nucleotides in a sequence.

[0215] As used herein the term “a polypeptide sequence as set forth inSEQ ID NO.:4” encompasses the polypeptide sequence of SEQ ID NO.:4, andsequences substantially identical thereto, polypeptide sequenceshomologous SEQ ID NO.:4, and sequences substantially identical thereto,or fragments of any of the preceding sequences. Homologous polypeptidesequences refer to a polypeptide sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% homology to SEQID NO.:4. Homology may be determined using any of the computer programsand parameters described herein, including FASTA version 3.0t78 with thedefault parameters or with any modified parameters. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. The polypeptidefragments comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,or 150 consecutive amino acids of the polypeptide of SEQ ID NO.:4, andsequences substantially identical thereto. It will be appreciated thatthe polypeptide codes as set forth in SEQ ID NO.:4, and sequencessubstantially identical thereto, can be represented in the traditionalsingle character format or three letter format (See the inside backcover of Stryer, Lubert. Biochemistry 3Ed. W. H Freeman & Co., NewYork.) or in any other format which relates the identity of thepolypeptides in a sequence.

[0216] It will be appreciated by those skilled in the art that a nucleicacid sequence as set forth in SEQ ID NO.:3 and a polypeptide sequence asset forth in SEQ ID NO.:4 can be stored, recorded, and manipulated onany medium which can be read and accessed by a computer. As used herein,the words “recorded” and “stored” refer to a process for storinginformation on a computer medium. A skilled artisan can readily adoptany of the presently known methods for recording information on acomputer readable medium to generate manufactures comprising SEQ IDNO.:3, and sequences substantially identical thereto, SEQ ID NO.:4, andsequences substantially identical thereto. Another aspect of theinvention is a computer readable medium having recorded thereon at least2, 5, 10, 15, or 20 nucleic acid sequences as set forth in SEQ ID NO.:3,and sequences substantially identical thereto.

[0217] Another aspect of the invention is a computer readable mediumhaving recorded thereon SEQ ID NO.:3, and sequences substantiallyidentical thereto. Another aspect of the invention is a computerreadable medium having recorded thereon SEQ ID NO.:4, and sequencessubstantially identical thereto. Another aspect of the invention is acomputer readable medium having recorded thereon at least 2, 5, 10, 15,or 20 of the sequences as set forth above.

[0218] Computer readable media include magnetically readable media,optically readable media, electronically readable media andmagnetic/optical media. For example, the computer readable media may bea hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital VersatileDisk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) aswell as other types of other media known to those skilled in the art.

[0219] Embodiments of the invention include systems (e.g., internetbased systems), particularly computer systems which store and manipulatethe sequence information described herein. One example of a computersystem 100 is illustrated in block diagram form in FIG. 1. As usedherein, “a computer system” refers to the hardware components, softwarecomponents, and data storage components used to analyze a nucleotidesequence of a nucleic acid sequence as set forth in SEQ ID NO.:3, andsequences substantially identical thereto, or a polypeptide sequence asset forth in SEQ ID NO.:4. The computer system 100 typically includes aprocessor for processing, accessing and manipulating the sequence data.The processor 105 can be any well-known type of central processing unit,such as, for example, the Pentium III from Intel Corporation, or similarprocessor from Sun, Motorola, Compaq, AMD or International BusinessMachines.

[0220] Typically the computer system 100 is a general purpose systemthat comprises the processor 105 and one or more internal data storagecomponents 110 for storing data, and one or more data retrieving devicesfor retrieving the data stored on the data storage components. A skilledartisan can readily appreciate that any one of the currently availablecomputer systems are suitable.

[0221] In one particular embodiment, the computer system 100 includes aprocessor 105 connected to a bus which is connected to a main memory 115(preferably implemented as RAM) and one or more internal data storagedevices 110, such as a hard drive and/or other computer readable mediahaving data recorded thereon. In some embodiments, the computer system100 further includes one or more data retrieving device 118 for readingthe data stored on the internal data storage devices 110.

[0222] The data retrieving device 118 may represent, for example, afloppy disk drive, a compact disk drive, a magnetic tape drive, or amodem capable of connection to a remote data storage system (e.g., viathe internet) etc. In some embodiments, the internal data storage device110 is a removable computer readable medium such as a floppy disk, acompact disk, a magnetic tape, etc. containing control logic and/or datarecorded thereon. The computer system 100 may advantageously include orbe programmed by appropriate software for reading the control logicand/or the data from the data storage component once inserted in thedata retrieving device.

[0223] The computer system 100 includes a display 120 which is used todisplay output to a computer user. It should also be noted that thecomputer system 100 can be linked to other computer systems 125 a-c in anetwork or wide area network to provide centralized access to thecomputer system 100.

[0224] Software for accessing and processing the nucleotide sequences ofa nucleic acid sequence as set forth in SEQ ID NO.:3, and sequencessubstantially identical thereto, or a polypeptide sequence as set forthin SEQ ID NO.:4, and sequences substantially identical thereto, (such assearch tools, compare tools, and modeling tools etc.) may reside in mainmemory 115 during execution.

[0225] In some embodiments, the computer system 100 may further comprisea sequence comparison algorithm for comparing a nucleic acid sequence asset forth in SEQ ID NO.:3, and sequences substantially identicalthereto, or a polypeptide sequence as set forth in SEQ ID NO.:4, andsequences substantially identical thereto, stored on a computer readablemedium to a reference nucleotide or polypeptide sequence(s) stored on acomputer readable medium. A “sequence comparison algorithm” refers toone or more programs which are implemented (locally or remotely) on thecomputer system 100 to compare a nucleotide sequence with othernucleotide sequences and/or compounds stored within a data storagemeans. For example, the sequence comparison algorithm may compare thenucleotide sequences of a nucleic acid sequence as set forth in SEQ IDNO.:3, and sequences substantially identical thereto, or a polypeptidesequence as set forth in SEQ ID NO.:4, and sequences substantiallyidentical thereto, stored on a computer readable medium to referencesequences stored on a computer readable medium to identify homologies orstructural motifs. Various sequence comparison programs identifiedelsewhere in this patent specification are particularly contemplated foruse in this aspect of the invention. Protein and/or nucleic acidsequence homologies may be evaluated using any of the variety ofsequence comparison algorithms and programs known in the art. Suchalgorithms and programs include, but are by no means limited to,TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc.Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al., J. Mol.Biol. 215(3):403-410, 1990; Thompson et al., Nucleic Acids Res.22(2):4673-4680, 1994; Higgins et al., Methods Enzymol. 266:383-402,1996; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Altschul etal., Nature Genetics 3:266-272, 1993).

[0226] Homology or identity is often measured using sequence analysissoftware (e.g., Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705). Such software matches similarsequences by assigning degrees of homology to various deletions,substitutions and other modifications. The terms “homology” and“identity” in the context of two or more nucleic acids or polypeptidesequences, refer to two or more sequences or subsequences that are thesame or have a specified percentage of amino acid residues ornucleotides that are the same when compared and aligned for maximumcorrespondence over a comparison window or designated region as measuredusing any number of sequence comparison algorithms or by manualalignment and visual inspection.

[0227] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Defaultprogram parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

[0228] A “comparison window”, as used herein, includes reference to asegment of any one of the number of contiguous positions selected fromthe group consisting of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequence for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol 48:443,1970, by the search for similarity method of person & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection. Otheralgorithms for determining homology or identity include, for example, inaddition to a BLAST program (Basic Local Alignment Search Tool at theNational Center for Biological Information), ALIGN, AMAS (Analysis ofMultiply Aligned Sequences), AMPS (Protein Multiple Sequence Alignment),ASSET (Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR,BIOSCAN (Biological Sequence Comparative Analysis Node), BLIMPS (BLocksIMProved Searcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTALW, CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN,Las Vegas algorithm, FNAT (Forced Nucleotide Alignment Tool),Framealign, Framesearch, DYNAMIC, FILTER, FSAP (Fristensky SequenceAnalysis Package), GAP (Global Alignment Program), GENAL, GIBBS,GenQuest, ISSC (Sensitive Sequence Comparison), LALIGN (Local SequenceAlignment), LCP (Local Content Program), MACAW (Multiple AlignmentConstruction & Analysis Workbench), MAP (Multiple Alignment Program),MBLKP, MBLKN, PIMA (Pattern-Induced Multi-sequence Alignment), SAGA(Sequence Alignment by Genetic Algorithm) and WHAT-IF. Such alignmentprograms can also be used to screen genome databases to identifypolynucleotide sequences having substantially identical sequences. Anumber of genome databases are available, for example, a substantialportion of the human genome is available as part of the Human GenomeSequencing Project (J. Roach,http://weber.u.Washington.edu/˜roach/human_genome_progress 2.html)(Gibbs, 1995). At least twenty-one other genomes have already beensequenced, including, for example, M. genitalium (Fraser et al., 1995),M. jannaschii (Bult et al., 1996), H. influenzae (Fleischmann et al.,1995), E. coli (Blattner et al., 1997), and yeast (S. cerevisiae) (Meweset al., 1997), and D. melanogaster (Adams et al., 2000). Significantprogress has also been made in sequencing the genomes of model organism,such as mouse, C. elegans, and Arabadopsis sp. Several databasescontaining genomic information annotated with some functionalinformation are maintained by different organization, and are accessiblevia the internet, for example, http://wwwtigr.org/tdb;http://www.genetics.wisc.edu; http://genome-www.stanford.edu/˜ball;http://hiv-web.lanl.gov; http://www.ncbi.nlm.nih.gov;http://www.ebi.ac.uk; http://Pasteur.fr/other/biology; andhttp://www.genome.wi.mit.edu.

[0229] One example of a useful algorithm is BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402, 1977, andAltschul etal., J.Mol.Biol.215:403-410,1990,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when:the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

[0230] The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Natl. Acad. Sci. USA 90:5873, 1993). One measure of similarity providedby BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a references sequence if thesmallest sum probability in a comparison of the test nucleic acid to thereference nucleic acid is less than about 0.2, more preferably less thanabout 0.01, and most preferably less than about 0.001.

[0231] In one embodiment, protein and nucleic acid sequence homologiesare evaluated using the Basic Local Alignment Search Tool (“BLAST”) Inparticular, five specific BLAST programs are used to perform thefollowing task:

[0232] (1) BLASTP and BLAST3 compare an amino acid query sequenceagainst a protein sequence database;

[0233] (2) BLASTN compares a nucleotide query sequence against anucleotide sequence database;

[0234] (3) BLASTX compares the six-frame conceptual translation productsof a query nucleotide sequence (both strands) against a protein sequencedatabase;

[0235] (4) TBLASTN compares a query protein sequence against anucleotide sequence database translated in all six reading frames (bothstrands); and

[0236] (5) TBLASTX compares the six-frame translations of a nucleotidequery sequence against the six-frame translations of a nucleotidesequence database.

[0237] The BLAST programs identify homologous sequences by identifyingsimilar segments, which are referred to herein as “high-scoring segmentpairs,” between a query amino or nucleic acid sequence and a testsequence which is preferably obtained from a protein or nucleic acidsequence database. High-scoring segment pairs are preferably identified(i.e., aligned) by means of a scoring matrix, many of which are known inthe art. Preferably, the scoring matrix used is the BLOSUM62 matrix(Gonnet et al., Science 256:1443-1445, 1992; Henikoff and Henikoff,Proteins 17:49-61, 1993). Less preferably, the PAM or PAM250 matricesmay also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matricesfor Detecting Distance Relationships:Atlas ofprotein Sequence andStructure, Washington: National Biomedical Research Foundation). BLASTprograms are accessible through the U.S. National Library of Medicine,e.g., at www.ncbi.nlm.nih.gov.

[0238] The parameters used with the above algorithms may be adapteddepending on the sequence length and degree of homology studied. In someembodiments, the parameters may be the default parameters used by thealgorithms in the absence of instructions from the user.

[0239]FIG. 2 is a flow diagram illustrating one embodiment of a process200 for comparing a new nucleotide or protein sequence with a databaseof sequences in order to determine the homology levels between the newsequence and the sequences in the database. The database of sequencescan be a private database stored within the computer system 100, or apublic database such as GENBANK that is available through the Internet.

[0240] The process 200 begins at a start state 201 and then moves to astate 202 wherein the new sequence to be compared is stored to a memoryin a computer system 100. As discussed above, the memory could be anytype of memory, including RAM or an internal storage device.

[0241] The process 200 then moves to a state 204 wherein a database ofsequences is opened for analysis and comparison. The process 200 thenmoves to a state 206 wherein the first sequence stored in the databaseis read into a memory on the computer. A comparison is then performed ata state 210 to determine if the first sequence is the same as the secondsequence. It is important to note that this step is not limited toperforming an exact comparison between the new sequence and the firstsequence in the database. Well-known methods are known to those of skillin the art for comparing two nucleotide or protein sequences, even ifthey are not identical. For example, gaps can be introduced into onesequence in order to raise the homology level between the two testedsequences. The parameters that control whether gaps or other featuresare introduced into a sequence during comparison are normally entered bythe user of the computer system.

[0242] Once a comparison of the two sequences has been performed at thestate 210, a determination is made at a decision state 210 whether thetwo sequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200.

[0243] If a determination is made that the two sequences are the same,the process 200 moves to a state 214 wherein the name of the sequencefrom the database is displayed to the user. This state notifies the userthat the sequence with the displayed name fulfills the homologyconstraints that were entered. Once the name of the stored sequence isdisplayed to the user, the process 200 moves to a decision state 218wherein a determination is made whether more sequences exist in thedatabase. If no more sequences exist in the database, then the process200 terminates at an end state 220. However, if more sequences do existin the database, then the process 200 moves to a state 224 wherein apointer is moved to the next sequence in the database so that it can becompared to the new sequence. In this manner, the new sequence isaligned and compared with every sequence in the database.

[0244] It should be noted that if a determination had been made at thedecision state 212 that the sequences were not homologous, then theprocess 200 would move immediately to the decision state 218 in order todetermine if any other sequences were available in the database forcomparison.

[0245] Accordingly, one aspect of the invention is a computer systemcomprising a processor, a data storage device having stored thereon anucleic acid sequence as set forth in SEQ ID NO.:3, and sequencessubstantially identical thereto, or a polypeptide sequence as set forthin SEQ ID NO.:4, and sequences substantially identical thereto, a datastorage device having retrievably stored thereon reference nucleotidesequences or polypeptide sequences to be compared to a nucleic acidsequence as set forth in SEQ ID NO.:3, and sequences substantiallyidentical thereto, or a polypeptide sequence as set forth in SEQ IDNO.:4, and sequences substantially identical thereto, and a sequencecomparer for conducting the comparison. The sequence comparer mayindicate a homology level between the sequences compared or identifystructural motifs in the above described nucleic acid code of SEQ IDNO.: 3, and sequences substantially identical thereto, or a polypeptidesequence as set forth in SEQ ID NO.:4, and sequences substantiallyidentical thereto, or it may identify structural motifs in sequenceswhich are compared to these nucleic acid codes and polypeptide codes. Insome embodiments, the data storage device may have stored thereon thesequences of at least 2, 5, 10, 15, 20, 25, 30 or 40 or more of thenucleic acid sequences as set forth in SEQ ID NO.:3, and sequencessubstantially identical thereto, or the polypeptide sequences as setforth in SEQ ID NO.:4, and sequences substantially identical thereto.

[0246] Another aspect of the invention is a method for determining thelevel of homology between SEQ ID NO.:3, and sequences substantiallyidentical thereto, or SEQ ID NO.:4, and sequences substantiallyidentical thereto, and a reference nucleotide sequence. The methodincluding reading the nucleic acid code or the polypeptide code and thereference nucleotide or polypeptide sequence through the use of acomputer program which determines homology levels and determininghomology between the nucleic acid code or polypeptide code and thereference nucleotide or polypeptide sequence with the computer program.The computer program may be any of a number of computer programs fordetermining homology levels, including those specifically enumeratedherein, (e.g., BLAST2N with the default parameters or with any modifiedparameters). The method may be implemented using the computer systemsdescribed above. The method may also be performed by reading at least 2,5, 10, 15, 20, 25, 30 or 40 or more of the above described nucleic acidsequences as set forth in SEQ ID NO.:3, or the polypeptide sequences asset forth in the SEQ ID NO.:4 through use of the computer program anddetermining homology between the nucleic acid codes or polypeptide codesand reference nucleotide sequences or polypeptide sequences.

[0247]FIG. 3 is a flow diagram illustrating one embodiment of a process250 in a computer for determining whether two sequences are homologous.The process 250 begins at a start state 252 and then moves to a state254 wherein a first sequence to be compared is stored to a memory. Thesecond sequence to be compared is then stored to a memory at a state256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it is preferably in the single letter amino acidcode so that the first and sequence sequences can be easily compared.

[0248] A determination is then made at a decision state 264 whether thetwo characters are the same. If they are the same, then the process 250moves to a state 268 wherein the next characters in the first and secondsequences are read. A determination is then made whether the nextcharacters are the same. If they are, then the process 250 continuesthis loop until two characters are not the same. If a determination ismade that the next two characters are not the same, the process 250moves to a decision state 274 to determine whether there are any morecharacters either sequence to read.

[0249] If there are not any more characters to read, then the process250 moves to a state 276 wherein the level of homology between the firstand second sequences is displayed to the user. The level of homology isdetermined by calculating the proportion of characters between thesequences that were the same out of the total number of sequences in thefirst sequence. Thus, if every character in a first 100 nucleotidesequence aligned with a every character in a second sequence, thehomology level would be 100%.

[0250] Alternatively, the computer program may be a computer programwhich compares the nucleotide sequences of a nucleic acid sequence asset forth in the invention, to one or more reference nucleotidesequences in order to determine whether the nucleic acid code of SEQ IDNO.:3, and sequences substantially identical thereto, differs from areference nucleic acid sequence at one or more positions. Optionallysuch a program records the length and identity of inserted, deleted orsubstituted nucleotides with respect to the sequence of either thereference polynucleotide or a nucleic acid sequence as set forth in SEQID NO.:3, and sequences substantially identical thereto. In oneembodiment, the computer program may be a program which determineswhether a nucleic acid sequence as set forth in SEQ ID NO.:3, andsequences substantially identical thereto, contains a single nucleotidepolymorphism (SNP) with respect to a reference nucleotide sequence.

[0251] Accordingly, another aspect of the invention is a method fordetermining whether a nucleic acid sequence as set forth in SEQ IDNO.:3, and sequences substantially identical thereto, differs at one ormore nucleotides from a reference nucleotide sequence comprising thesteps of reading the nucleic acid code and the reference nucleotidesequence through use of a computer program which identifies differencesbetween nucleic acid sequences and identifying differences between thenucleic acid code and the reference nucleotide sequence with thecomputer program. In some embodiments, the computer program is a programwhich identifies single nucleotide polymorphisms. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 3. The method may also be performed by reading atleast 2, 5, 10, 15, 20, 25, 30, or40 or more of the nucleic acidsequences as set forth in SEQ ID NO.:3, and sequences substantiallyidentical thereto, and the reference nucleotide sequences through theuse of the computer program and identifying differences between thenucleic acid codes and the reference nucleotide sequences with thecomputer program.

[0252] In other embodiments the computer based system may furthercomprise an identifier for identifying features within a nucleic acidsequence as set forth in SEQ ID NO.:3 or a polypeptide sequence as setforth in SEQ ID NO.:4, and sequences substantially identical thereto.

[0253] An “identifier” refers to one or more programs which identifiescertain features within a nucleic acid sequence as set forth in SEQ IDNO.:3, and sequences substantially identical thereto, or a polypeptidesequence as set forth in SEQ ID NO.:4, and sequences substantiallyidentical thereto. In one embodiment, the identifier may comprise aprogram which identifies an open reading frame in a nucleic acidsequence as set forth in SEQ ID NO.:3, and sequences substantiallyidentical thereto.

[0254]FIG. 5 is a flow diagram illustrating one embodiment of anidentifier process 300 for detecting the presence of a feature in asequence. The process 300 begins at a start state 302 and then moves toa state 304 wherein a first sequence that is to be checked for featuresis stored to a memory 115 in the computer system 100. The process 300then moves to a state 306 wherein a database of sequence features isopened. Such a database would include a list of each feature'sattributes along with the name of the feature. For example, a featurename could be “Initiation Codon” and the attribute would be “ATG”.Another example would be the feature name “TAATAA Box” and the featureattribute would be “TAATAA”. An example of such a database is producedby the University of Wisconsin Genetics Computer Group (www.gcg.com).Alternatively, the features may be structural polypeptide motifs such asalpha helices, beta sheets, or functional polypeptide motifs such asenzymatic active sites, helix-turn-helix motifs or other motifs known tothose skilled in the art.

[0255] Once the database of features is opened at the state 306, theprocess 300 moves to a state 308 wherein the first feature is read fromthe database. A comparison of the attribute of the first feature withthe first sequence is then made at a state 310. A determination is thenmade at a decision state 316 whether the attribute of the feature wasfound in the first sequence. If the attribute was found, then theprocess 300 moves to a state 318 wherein the name of the found featureis displayed to the user.

[0256] The process 300 then moves to a decision state 320 wherein adetermination is made whether move features exist in the database. If nomore features do exist, then the process 300 terminates at an end state324. However, if more features do exist in the database, then theprocess 300 reads the next sequence feature at a state 326 and loopsback to the state 310 wherein the attribute of the next feature iscompared against the first sequence.

[0257] It should be noted, that if the feature attribute is not found inthe first sequence at the decision state 316, the process 300 movesdirectly to the decision state 320 in order to determine if any morefeatures exist in the database.

[0258] Accordingly, another aspect of the invention is a method ofidentifying a feature within a nucleic acid sequence as set forth in SEQID NO.:3, and sequences substantially identical thereto, or apolypeptide sequence as set forth in SEQ ID NO.:4, and sequencessubstantially identical thereto, comprising reading the nucleic acidcode(s) or polypeptide code(s) through the use of a computer programwhich identifies features therein and identifying features within thenucleic acid code(s) with the computer program. In one embodiment,computer program comprises a computer program which identifies openreading frames. The method may be performed by reading a single sequenceor at least 2, 5, 10, 15, 20, 25, 30, or 40 of the nucleic acidsequences as set forth in SEQ ID NO.:3, and sequences substantiallyidentical thereto, or the polypeptide sequences as set forth in SEQ IDNO.:4, and sequences substantially identical thereto, through the use ofthe computer program and identifying features within the nucleic acidcodes or polypeptide codes with the computer program.

[0259] A nucleic acid sequence as set forth in SEQ ID NO.:3, andsequences substantially identical thereto, or a polypeptide sequence asset forth in SEQ ID NO.:4, and sequences substantially identicalthereto, may be stored and manipulated in a variety of data processorprograms in a variety of formats. For example, a nucleic acid sequenceas set forth in SEQ ID NO.:3, and sequences substantially identicalthereto, or a polypeptide sequence as set forth in SEQ ID NO.:4, andsequences substantially identical thereto, may be stored as text in aword processing file, such as MicrosoftWORD or WORDPERFECT or as anASCII file in a variety of database programs familiar to those of skillin the art, such as DB2, SYBASE, or ORACLE. In addition, many computerprograms and databases may be used as sequence comparison algorithms,identifiers, or sources of reference nucleotide sequences or polypeptidesequences to be compared to a nucleic acid sequence as set forth in SEQID NO.:3, and sequences substantially identical thereto, or apolypeptide sequence as set forth in SEQ ID NO.:4, and sequencessubstantially identical thereto. The following list is intended not tolimit the invention but to provide guidance to programs and databaseswhich are useful with the nucleic acid sequences as set forth in SEQ IDNO.:3, and sequences substantially identical thereto, or the polypeptidesequences as set forth in SEQ ID NO.:4, and sequences substantiallyidentical thereto.

[0260] The programs and databases which may be used include, but are notlimited to:MacPattern (EMBL), DiscoveryBase (Molecular ApplicationsGroup), GeneMine (Molecular Applications Group), Look (MolecularApplications Group), MacLook (Molecular Applications Group), BLAST andBLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215:403,1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444,1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990),Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (MolecularSimulations Inc.), Cerius².DBAccess (Molecular Simulations Inc.),HypoGen (Molecular Simulations Inc.), Insight II, (Molecular SimulationsInc.), Discover (Molecular Simulations Inc.), CHARMm (MolecularSimulations Inc.), Felix (Molecular Simulations Inc.), DelPhi,(Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.),Homology (Molecular Simulations Inc.), Modeler (Molecular SimulationsInc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design(Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.),WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer(Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), theMDL Available Chemicals Directory database, the MDL Drug Data Reportdata base, the Comprehensive Medicinal Chemistry database, Derwent'sWorld Drug Index database, the BioByteMasterFile database, the Genbankdatabase, and the Genseqn database. Many other programs and data baseswould be apparent to one of skill in the art given the presentdisclosure.

[0261] Motifs which may be detected using the above programs includesequences encoding leucine zippers, helix-turn-helix motifs,glycosylation sites, ubiquitination sites, alpha helices, and betasheets, signal sequences encoding signal peptides which direct thesecretion of the encoded proteins, sequences implicated in transcriptionregulation such as homeoboxes, acidic stretches, enzymatic active sites,substrate binding sites, and enzymatic cleavage sites.

[0262] The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds, such assmall molecules. Each biocatalyst is specific for one functional group,or several related functional groups, and can react with many startingcompounds containing this functional group.

[0263] The biocatalytic reactions produce a population of derivativesfrom a single starting compound. These derivatives can be subjected toanother round of biocatalytic reactions to produce a second populationof derivative compounds. Thousands of variations of the original smallmolecule or compound can be produced with each iteration of biocatalyticderivatization.

[0264] Enzymes react at specific sites of a starting compound withoutaffecting the rest of the molecule, a process which is very difficult toachieve using traditional chemical methods. This high degree ofbiocatalytic specificity provides the means to identify a single activecompound within the library. The library is characterized by the seriesof biocatalytic reactions used to produce it, a so called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies, and compounds can besynthesized and tested free in solution using virtually any type ofscreening assay. It is important to note, that the high degree ofspecificity of enzyme reactions on functional groups allows for the“tracking” of specific enzymatic reactions that make up thebiocatalytically produced library.

[0265] Many of the procedural steps are performed using roboticautomation enabling the execution of many thousands of biocatalyticreactions and screening assays per day as well as ensuring a high levelof accuracy and reproducibility. As a result, a library of derivativecompounds can be produced in a matter of weeks which would take years toproduce using current chemical methods.

[0266] In a particular embodiment, the invention provides a method formodifying small molecules, comprising contacting a polypeptide encodedby a polynucleotide described herein or enzymatically active fragmentsthereof with a small molecule to produce a modified small molecule. Alibrary of modified small molecules is tested to determine if a modifiedsmall molecule is present within the library which exhibits a desiredactivity. A specific biocatalytic reaction which produces the modifiedsmall molecule of desired activity is identified by systematicallyeliminating each of the biocatalytic reactions used to produce a portionof the library, and then testing the small molecules produced in theportion of the library for the presence or absence of the modified smallmolecule with the desired activity. The specific biocatalytic reactionswhich produce the modified small molecule of desired activity isoptionally repeated. The biocatalytic reactions are conducted with agroup of biocatalysts that react with distinct structural moieties foundwithin the structure of a small molecule, each biocatalyst is specificfor one structural moiety or a group of related structural moieties; andeach biocatalyst reacts with many different small molecules whichcontain the distinct structural moiety.

[0267] The invention will be further described with reference to thefollowing examples; however, it is to be understood that the inventionis not limited to such examples.

EXAMPLES Example 1

[0268] Production of the Expression Gene Bank

[0269] Colonies containing pBluescript plasmids with random inserts fromthe organism Thermococcus alcaliphilus AEDII12RA were obtained from anoriginal XZAP2 genomic library generated according to the manufacturer's(Stratagene) protocol. The clones were then excised from λZAP2 topBluescript. The clones were excised to pBluescript according to themethod of Hay and Short. (Hay, B. and Short, J. Strategies, 1992, 5:16.)The resulting colonies were picked with sterile toothpicks and used tosingly inoculate each of the wells of 96-well microtiter plates. Thewells contained 250 μL of LB media with 100 μg/ml methicillin, and 10%v/v glycerol (LB Amp/Meth, glycerol). The cells were grown overnight at37° C. without shaking. This constituted generation of the “SourceGeneBank”; each well of the Source GeneBank thus contained a stockculture of E. coli cells, each of which contained a pBluescript plasmidwith a unique DNA insert.

Example 2

[0270] Screening for Glycosidase Activity

[0271] The plates of the Source GeneBank were used to multiply inoculatea single plate (the “Condensed Plate”) containing in each well 200 μL ofLB Amp/Meth, glycerol. This step was performed using the High DensityReplicating Tool (HDRT) of the Beckman Biomek with a 1% bleach, water,isopropanol, air-dry sterilization cycle in between each inoculation.Each well of the Condensed Plate thus contained 10 to 12 differentpBluescript clones from each of the source library plates. The CondensedPlate was grown for 16 h at 37° C. and then used to inoculate two white96-well Polyfiltronics microtiter daughter plates containing in eachwell 250 μL of LB Amp/Meth (without glycerol). The original condensedplate was put in storage −80° C. The two condensed daughter plates wereincubated at 37° C. for 18 h.

[0272] A ‘600 μM substrate stock solution’ was prepared as follows:25 mgof each of four compounds was dissolved in the appropriate volume ofDMSO to yield a 25.2 mM solution. The compounds used were4-methylumbelliferyl β-D-xyloside, 4-methylumbelliferyl α-D-galactoside,4-methylumbelliferyl α-D-mannopyranoside, and 4-methylumbelliferylβ-D-mannopyranoside. Two hundred fifty microliters of each DMSO solutionwas added to ca. 9 mL of 50 mM, pH 7.5 Hepes buffer. The volume wastaken to 10.5 mL with the above Hepes buffer to yield a clear solution.All four umbelliferones were obtained from Sigma Chemical Co.

[0273] Fifty μL of the ‘600 μM stock solution’ was added to each of thewells of a white condensed plate using the Biomek to yield a finalconcentration of substrate of ˜100 μM. The fluorescence values wererecorded (excitation=326 nm, emission=450 nm) on a plate readingfluorometer immediately after addition of the substrate. The plate wasincubated at 70° C. for 60 min and the fluorescence values were recordedagain. The initial and final fluorescence values were subtracted todetermine if an active clone was present by an increase in fluorescenceover the majority of the other wells.

Example 3

[0274] Isolation of Active Clone and Substrate Specificity Determination

[0275] In order to isolate the individual clone which carried theactivity, the Source GeneBank plates were thawed and the individualwells used to singly inoculate a new plate containing LB/Amp/Meth. Asabove the plate was incubated at 37° C. to grow the cells, the 50 μL of600 μM substrate stock solution added using the Biomek. Once the activewell from the source plate was identified, the cells from the sourceplate were streaked on agar with LB/Amp/Meth and grown overnight at to37° C. to obtain single colonies. Eight single colonies were picked witha sterile toothpick and used to singly inoculate the wells of a 96-wellmicrotiter plate. The wells contained 250 μL of LB/Amp/Meth. The cellswere grown overnight at 37° C. without shaking. A 200 μL aliquot wasremoved from each well and assayed with the substrates as above. Themost active clone was identified and the remaining 50 μL of culture wasused to streak an agar plate with LB/Amp/Meth. Eight single colonieswere picked, grown and assayed as above. The most active clone was usedto inoculate 3 mL cultures of LB/Amp/Meth, which were grown overnight.The plasmid DNA was isolated from the cultures and utilized forsequencing. Colonies from this final streak onto the agar plate werealso used to inoculate wells containing 250 μL of LB/Amp/Meth. Inaddition, colonies containing plasmids with no inserts were used asnegative controls. A 600 μM solution of each individual substrate wasmade up for the purpose of determining the substrate specificity of theenzyme. Fifty μL of each of the four substrates were added individuallyto the test and control wells and assayed for activity as above. Onlythe wells which contained the 4-methylumbelliferyl α-D-galactosideshowed an increase in fluorescence indicating activity.

[0276] While the invention has been described in detail with referenceto certain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

1 4 1 52 DNA Artificial Sequence polynucleotide probe 1 ccgagaattcattaaagagg agaaattaac tatgagagcg ctcgtctttc ac 52 2 31 DNA ArtificialSequence polynucleotide probe 2 cggaagatct aggttcccca ttttcacccc t 31 31095 DNA Thernococcus alcaliphilus CDS (1)...(1092) 3 ttg aga gcg ctcgtc ttt cac ggc aac ctc cag tat gcc gaa atc cca 48 Leu Arg Ala Leu ValPhe His Gly Asn Leu Gln Tyr Ala Glu Ile Pro 1 5 10 15 aag agc gaa atccca aag gtc ata gag aag gca tac atc cca gtc atc 96 Lys Ser Glu Ile ProLys Val Ile Glu Lys Ala Tyr Ile Pro Val Ile 20 25 30 gag aca ctg att aaagaa gaa att cct ttt ggg ctc aac ata acg ggc 144 Glu Thr Leu Ile Lys GluGlu Ile Pro Phe Gly Leu Asn Ile Thr Gly 35 40 45 tat acc tta aag ttc ctcccg aag gat att ata gac ctc gtt aaa ggg 192 Tyr Thr Leu Lys Phe Leu ProLys Asp Ile Ile Asp Leu Val Lys Gly 50 55 60 ggc atc gcg agt gac ctg atagag ata atc gga acg agc tac acg cac 240 Gly Ile Ala Ser Asp Leu Ile GluIle Ile Gly Thr Ser Tyr Thr His 65 70 75 80 gca ata ctc ccc ctc ctc ccgctt agc aga gta gaa gca caa gtt cag 288 Ala Ile Leu Pro Leu Leu Pro LeuSer Arg Val Glu Ala Gln Val Gln 85 90 95 aga gat agg gaa gtt aag gaa gagctc ttc gag ctt tct cca aag gga 336 Arg Asp Arg Glu Val Lys Glu Glu LeuPhe Glu Leu Ser Pro Lys Gly 100 105 110 ttc tgg ctg cca gag ctc gcc tatgac ccg ata atc cct gcc ata ctg 384 Phe Trp Leu Pro Glu Leu Ala Tyr AspPro Ile Ile Pro Ala Ile Leu 115 120 125 aag gac aac ggt tat gag tat ctattc gcc gac ggg gag gcg atg ctt 432 Lys Asp Asn Gly Tyr Glu Tyr Leu PheAla Asp Gly Glu Ala Met Leu 130 135 140 ttc tca gct cat ctc aac tcg gcgata aag cca att aaa ccg ctc tat 480 Phe Ser Ala His Leu Asn Ser Ala IleLys Pro Ile Lys Pro Leu Tyr 145 150 155 160 cca cac ctt ata aag gcc caaagg gaa aag cgc ttt agg tac atc agc 528 Pro His Leu Ile Lys Ala Gln ArgGlu Lys Arg Phe Arg Tyr Ile Ser 165 170 175 tat ctc ctt ggt ctc agg gagctt agg aag gcg ata aag ctc gtt ttt 576 Tyr Leu Leu Gly Leu Arg Glu LeuArg Lys Ala Ile Lys Leu Val Phe 180 185 190 gaa ggt aag gta acg cta aaggca gtc aaa gac atc gaa gcc gta ccc 624 Glu Gly Lys Val Thr Leu Lys AlaVal Lys Asp Ile Glu Ala Val Pro 195 200 205 gtt tgg gtg gcc gtg aac acggct gta atg ctc ggc atc gga agg ctt 672 Val Trp Val Ala Val Asn Thr AlaVal Met Leu Gly Ile Gly Arg Leu 210 215 220 cct ctt atg aat cct aag aaagtg gcg agc tgg ata gag gac aag gac 720 Pro Leu Met Asn Pro Lys Lys ValAla Ser Trp Ile Glu Asp Lys Asp 225 230 235 240 aac att ctt cta tac ggcacc gat ata gag ttc att ggc tat agg gac 768 Asn Ile Leu Leu Tyr Gly ThrAsp Ile Glu Phe Ile Gly Tyr Arg Asp 245 250 255 att gca ggc tac aga atgagt gtt gag gga tta tta gag gtt ata gac 816 Ile Ala Gly Tyr Arg Met SerVal Glu Gly Leu Leu Glu Val Ile Asp 260 265 270 gag ctc aac tcg gaa ctgtgc ctt ccc tca gag ctg aag cac agt gga 864 Glu Leu Asn Ser Glu Leu CysLeu Pro Ser Glu Leu Lys His Ser Gly 275 280 285 agg gag ctc tac tta cggact tcg agt tgg gca cca gat aag agc ttg 912 Arg Glu Leu Tyr Leu Arg ThrSer Ser Trp Ala Pro Asp Lys Ser Leu 290 295 300 agg ata tgg aga gag gacgaa ggg aac gca aga ctt aat atg ctg tcc 960 Arg Ile Trp Arg Glu Asp GluGly Asn Ala Arg Leu Asn Met Leu Ser 305 310 315 320 tac aat atg agg ggcgaa ctc gcc ctt tta gcc gag aac agc gat gca 1008 Tyr Asn Met Arg Gly GluLeu Ala Leu Leu Ala Glu Asn Ser Asp Ala 325 330 335 agg gga tgg gag cccctc cct gag agg agg ctg gat gcc ttc cgg gcg 1056 Arg Gly Trp Glu Pro LeuPro Glu Arg Arg Leu Asp Ala Phe Arg Ala 340 345 350 ata tat aac gat tggagg ggt gaa aat ggg gaa cct tag 1095 Ile Tyr Asn Asp Trp Arg Gly Glu AsnGly Glu Pro 355 360 4 364 PRT Thermococcus alcaliphilus 4 Leu Arg AlaLeu Val Phe His Gly Asn Leu Gln Tyr Ala Glu Ile Pro 1 5 10 15 Lys SerGlu Ile Pro Lys Val Ile Glu Lys Ala Tyr Ile Pro Val Ile 20 25 30 Glu ThrLeu Ile Lys Glu Glu Ile Pro Phe Gly Leu Asn Ile Thr Gly 35 40 45 Tyr ThrLeu Lys Phe Leu Pro Lys Asp Ile Ile Asp Leu Val Lys Gly 50 55 60 Gly IleAla Ser Asp Leu Ile Glu Ile Ile Gly Thr Ser Tyr Thr His 65 70 75 80 AlaIle Leu Pro Leu Leu Pro Leu Ser Arg Val Glu Ala Gln Val Gln 85 90 95 ArgAsp Arg Glu Val Lys Glu Glu Leu Phe Glu Leu Ser Pro Lys Gly 100 105 110Phe Trp Leu Pro Glu Leu Ala Tyr Asp Pro Ile Ile Pro Ala Ile Leu 115 120125 Lys Asp Asn Gly Tyr Glu Tyr Leu Phe Ala Asp Gly Glu Ala Met Leu 130135 140 Phe Ser Ala His Leu Asn Ser Ala Ile Lys Pro Ile Lys Pro Leu Tyr145 150 155 160 Pro His Leu Ile Lys Ala Gln Arg Glu Lys Arg Phe Arg TyrIle Ser 165 170 175 Tyr Leu Leu Gly Leu Arg Glu Leu Arg Lys Ala Ile LysLeu Val Phe 180 185 190 Glu Gly Lys Val Thr Leu Lys Ala Val Lys Asp IleGlu Ala Val Pro 195 200 205 Val Trp Val Ala Val Asn Thr Ala Val Met LeuGly Ile Gly Arg Leu 210 215 220 Pro Leu Met Asn Pro Lys Lys Val Ala SerTrp Ile Glu Asp Lys Asp 225 230 235 240 Asn Ile Leu Leu Tyr Gly Thr AspIle Glu Phe Ile Gly Tyr Arg Asp 245 250 255 Ile Ala Gly Tyr Arg Met SerVal Glu Gly Leu Leu Glu Val Ile Asp 260 265 270 Glu Leu Asn Ser Glu LeuCys Leu Pro Ser Glu Leu Lys His Ser Gly 275 280 285 Arg Glu Leu Tyr LeuArg Thr Ser Ser Trp Ala Pro Asp Lys Ser Leu 290 295 300 Arg Ile Trp ArgGlu Asp Glu Gly Asn Ala Arg Leu Asn Met Leu Ser 305 310 315 320 Tyr AsnMet Arg Gly Glu Leu Ala Leu Leu Ala Glu Asn Ser Asp Ala 325 330 335 ArgGly Trp Glu Pro Leu Pro Glu Arg Arg Leu Asp Ala Phe Arg Ala 340 345 350Ile Tyr Asn Asp Trp Arg Gly Glu Asn Gly Glu Pro 355 360

What is claimed is:
 1. A computer readable medium having stored thereon a nucleic acid sequence as set forth in SEQ ID NO:3, and sequences substantially identical thereto, or a polypeptide sequence as set forth in SEQ ID NO:4, and sequences substantially identical thereto. 